Download Evidence for evolution of canine parvovirus type 2 in Italy

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of General Virology (2001), 82, 3021–3025. Printed in Great Britain
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SHORT COMMUNICATION
Evidence for evolution of canine parvovirus type 2 in Italy
Canio Buonavoglia,1 Vito Martella,1 Annamaria Pratelli,1 Maria Tempesta,1 Alessandra Cavalli,1
Domenico Buonavoglia,2 Giancarlo Bozzo,1 Gabriella Elia,1 Nicola Decaro1 and Leland Carmichael3
1
Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, S.p. per Casamassima km 3, 70010 Valenzano,
Bari, Italy
2
Department of Pathology and Infectious Diseases, University of Messina, Messina, Italy
3
James Baker Institute, Cornell University, Ithaca, NY, USA
Two isolates of canine parvovirus (CPV) were obtained from dogs affected with severe haemorrhagic diarrhoea. Type 2b antigenic specificity was
predicted by both antigenic analysis with monoclonal antibodies and PCR characterization with
type-specific primers. Nevertheless, sequence
analysis of the capsid protein-encoding gene revealed two amino acid changes. One of the changes
affected position 426 (Asp to Glu), in a major
antigenic site of the viral capsid, determining the
replacement of a residue unique to CPV type 2b.
The failure of established typing methods to distinguish this antigenic variant was overcome by the
development of an RFLP assay.
During the early 1970s, a new infectious disease of pups,
characterized by either gastroenteritis or myocarditis, was
observed worldwide. A small, round, non-enveloped virus was
observed by electron microscopy in stool specimens and in
tissues of affected animals. Subsequently, a novel parvovirus
was isolated both in canine and feline cell cultures (Kelly,
1978 ; Appel et al., 1979 ; Burtonboy et al., 1979 ; Johnson &
Spradbrow, 1979). The virus was named canine parvovirus
type 2 (CPV-2), to distinguish it from the previously described
parvovirus canine minute virus (CMV or CPV-1), which is
antigenically unrelated to CPV-2 (Carmichael & Binn,
1981 ; Carmichael et al., 1994).
CPV possesses a single-stranded DNA genome of negative
polarity, about 5200 nt in length. The CPV capsid is a 26 nm
diameter icosahedron made up of a combination of two
proteins, VP1 and VP2, formed by alternative splicing from the
same RNA (Reed et al., 1988). The mutation rate of the CPV
genome has not been determined ; however, since parvovirus
DNA is replicated by host cell DNA polymerases (Cotmore &
Author for correspondence : Canio Buonavoglia.
Fax j39 080 4679843. e-mail c.buonavoglia!veterinaria.uniba.it
0001-7880 # 2001 SGM
Tattersall, 1987), which have low error rates, mutational events
affecting the biological properties of parvoviruses presumably
occur very rarely. Estimates suggest that the rate of variation
is 1i10−% to 4i10−% changes\nt\year (Parrish, 1991 ;
Truyen et al., 1995).
In 1979 and 1980, an antigenic variant of CPV was
identified in several different countries by using monoclonal
antibodies (MAbs) and the variant was termed CPV type 2a
(Parrish et al., 1985, 1991). In the mid 1980s, the virus
underwent a further antigenic change, and the new variant was
referred to as CPV type 2b (Parrish et al., 1991). Currently, the
antigenic variants of CPV have completely replaced the
original type 2 and are variously distributed in canine
populations worldwide (Mochizuki et al., 1993 ; de Ybanez
et al., 1995 ; Greenwood et al., 1996 ; Truyen et al., 1996,
2000 ; Steinel et al., 1998 ; Sagazio et al., 1998 ; Buonavoglia et
al., 2000 ; Pereira et al., 2000).
While the antigenic variants differ from the original type in
five or six amino acids, the variant type 2b differs from type 2a
in only two positions, Asn-426 to Asp and Ile-555 to Val
(Parrish et al., 1991). Residue 426 is placed in a major antigenic
site (epitope A) over the three-fold spike of the capsid and the
mutation Asn-426 to Asp differentiates CPV-2b not only from
CPV types 2 and 2a, but also from feline parvovirus (FPV) and
mink enteritis virus (MEV). Conversely, residue 555 lies in a
minor antigenic site and the mutation of Ile-555 to Val
represents a reversion to or a retention of the sequence of the
original type 2 (Table 2) (Parrish et al., 1991 ; Agbandje et al.,
1995 ; Strassheim et al., 1994).
Following the onset and rapid spread of the antigenic
variants 2a and 2b, there has been little evidence of further
significant antigenic evolution : the analysis of CPV strains
isolated from either domestic or wild felines and canines has
demonstrated, with no or a few exceptions (Ikeda et al.,
2000 ; Steinel et al., 2000 ; Truyen et al., 2000), that, during the
last 15 years, both CPV types 2a and 2b have retained the
antigenic configuration defined originally by Parrish et al.
(1985, 1991).
This communication describes the isolation in Italy of two
unusual CPV strains collected during 2000. Both isolates were
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C. Buonavoglia and others
Table 1. Primers used for genotyping and sequence analysis
Primer*
P2sa
P2asa
Pabsb
Pabasb
Pbsb
Pbasb
Hforc
Hrevc
555forc
555revc
Sequence (5h
3h)
GAAGAGTGGTTGTAAATAATA
CCTATATCACCAAAGTTAGTAG
GAAGAGTGGTTGTAAATAATT
CCTATATAACCAAAGTTAGTAC
CTTTAACCTTCCTGTAACAG
CATAGTTAAATTGGTTATCTAC
CAGGTGATGAATTTGCTACA
CATTTGGATAAACTGGTGGT
CAGGAAGATATCCAGAAGGA
GGTGCTAGTTGATATGTAATAAACA
Sense
Position†
j
k
j
k
j
k
j
k
j
k
3025–3045
3706–3685
3025–3045
3706–3685
4043–4062
4470–4449
3556–3575
4185–4166
4003–4022
4585–4561
* Primers sequences were obtained from : a, Senda et al. (1995) ; b, Pereira et al. (2000) ; c, this study.
† Primer locations are indicated as reported in the corresponding papers. The positions of the primers
designed in this study are referred to strain CPV-b (accession no. M38245).
characterized as type 2b viruses by means of antigenic and
PCR analysis. However, analysis of the sequence of the capsid
protein-encoding gene revealed an amino acid change in a
strategic epitope of the capsid that differentiates both strains
from classical type 2b CPVs.
Two dogs, respectively 2 years (F56\00) and 2 months
(F136\00) old, were presented for clinical examination during
early 2000 with signs of severe haemorrhagic gastroenteritis.
Suspensions (10 %, w\v) of faecal samples from the animals
were positive for CPV by a haemagglutination (HA) test and
two isolates were made on A-72 cells grown in DMEM with
10 % foetal bovine serum. Virus growth on infected cells was
monitored by HA test and by an indirect immunofluorescence
test.
A panel of four MAbs (A4E3, B4A2, C1D1 and B4E1),
kindly supplied by C. R. Parrish (Cornell University, Ithaca,
NY, USA), was used for antigenic typing in a haemagglutination inhibition test. MAb analysis was performed both
on suspensions of faecal samples and on viruses after one, three
and five passages in cell culture.
In order to predict the antigenic type of strains 56\00 and
136\00, we also used a genotyping approach, based on the
PCR strategies reported by Pereira et al. (2000) and Senda et al.
(1995). Primer pairs Pabs\Pabas and P2s\P2as (Table 1),
recognizing the variants and the original type, respectively,
were selected in overlapping positions (3025–3045 for Pabs
and P2s, 3685–3706 for Pabas and P2as) ; the nucleotide
differences between the primers are restricted to one base in
the 3h end of each primer. Since the PCR products were of the
same size, the primers were used in separate sets of reactions.
Primer pair Pbs\Pbas (4043–4062 and 4449–4470 ; Table 1)
recognizes CPV type 2b selectively. Differential amplification
of CPV type 2b is ensured by single nucleotide variations in
the very 3h end of each primer and these nucleotide variations
(A G at 4062 and G A at 4449) correspond to the
DACC
strategic amino acid changes Asn-426 to Asp and Ile-555 to
Val that determine the antigenic variation from type 2a to type
2b. Therefore, CPV type 2a is recognized by the primer pair
Pabs\Pabas only, while CPV type 2b is recognized by both
primer pairs Pabs\Pabas and Pbs\Pbas. PCR analysis was
performed both on faecal samples and on viruses after one,
three and five passages in cell cultures.
The sequence was determined of a fragment of about 1100
nt, encoding a large region of the capsid protein of strains
56\00 and 136\00. Sequence analysis was performed on the
virus present in faecal samples and in the first, third and fifth
virus passage in A-72 cells. Also, the partial sequences were
determined of an additional eight CPV strains (Table 2)
isolated in Italy during 1998 and 1999. All these strains had
been characterized as type 2b by MAb analysis and by PCR
genotyping.
For sequence analysis, two primer pairs, Hfor\Hrev and
555for\555rev (Table 1), were selected to amplify a large
fragment of the capsid protein-encoding gene of CPV,
encompassing at least six or seven informative amino acids
responsible for important biological properties of the virus
(Parrish et al., 1991 ; Parker & Parrish, 1997 ; Chang et al., 1992).
The PCR products were purified and then sequenced directly
by using an ABI PRISM 377 sequencer (Perkin Elmer). For
sequence comparison, sequences of type 2, 2a and 2b CPVs
were selected from the databases.
The DNA sequences of strains 56\00 and 136\00 were
searched for matches to the recognition sites of a large
database of restriction enzymes by computer-aided analysis. A
restriction enzyme, MboII, was found to recognize selectively
the sequence GAGAA at nt 4062–4066, allowing differentiation of 56\00- and 136\00-like strains from CPV type 2, 2a
and 2b strains. Primer pair 555for\555rev was used to amplify
a 583 nt fragment that included the MboII restriction site. The
PCR product obtained was digested with 5 U MboII.
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Table 2. Amino acid variation in the VP2 capsid protein of the Italian CPV isolates
Amino acid residue
87
101
297
3674–3676
TCT (Ser)
GCT (Ala)
300
305
… Nucleotide positions
… Codons observed
Strain
555
Origin (year)
Accession no.
USA (1967)
M38246
Leu
Ile
Ser
Ala
Asp
Asp
Thr
Ile
Asn
Val
USA (1975)
M24001
Leu
Ile
Ser
Val
Asp
Asp
Thr
Ile
Asn
Val
USA (1978)
USA (1978)
M38245
M19296
Met
Met
Ile
Ile
Ser
Ser
Ala
Ala
Asp
Asp
Asn
Asp
Thr
Thr
Ile
Ile
Asn
Asn
Val
Val
USA (1984)
USA (1983)
M24003
M24000
Met
Met
Thr
Thr
Ser
Ser
Gly
Gly
Tyr
Tyr
Asp
Asp
Thr
Thr
Ile
Ile
Asn
Asn
Ile
Ile
USA (1984)
USA (1990)
Italy (1998)
Italy (1998)
Italy (1998)
Italy (1998)
Italy (1998)
Italy (1999)
Italy (1999)
M74849
M74852
–
–
–
–
–
–
–
Met
Met







Thr
Thr







Ser
Ser
Ala
Ala
Ala
Ala
Ala
Ala
Ser
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Asp
Asp
Asp
Asn
Asn
Asp
Asn
Asn
Asp
Thr
Thr
Thr
Asn
Asn
Thr
Asn
Asn
Thr
Ile
Ile
Ile
Thr
Thr
Ile
Thr
Ile
Ile
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Asp
Val
Val
Val
Val
Val
Val
Val
Val
Val
Italy (2000)
Italy (2000)
–
–




Ala
Ala
Gly
Gly
Tyr
Tyr
Asp
Asp
Thr
Thr
Ile
Ile
Glu
Glu
Val
Val
, Not determined.
DACD
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Natural mutants of canine parvovirus
FPV
FPV-b
MEV
MEV-b
CPV type 2
CPV-b
CPV-Norden
CPV type 2a
CPV-15
CPV-31
CPV type 2b
CPV-39
CPV-133
242\98
244\98
224\98
212\98
197\98
111\99
01\99
CPV type 2b ?
136\00
56\00
375
389
418
426
3909–3911 3951–3953 4038–4040 4062–4064
GAT (Asp)
ACC (Thr)
ATT (Ile)
AAT (Asn)
AAT (Asn)
AAC (Asn)
ACT (Thr)
GAT (Asp)
GAA (Glu)
C. Buonavoglia and others
Strains 56\00 and 136\00 were identified as type 2b by
both MAb analysis and PCR genotyping. Nevertheless, in
sequence comparisons with well-established type 2, 2a and 2b
strains, strains 56\00 and 136\00 were found to contain two
unexpected amino acids variations, Ser-297 to Ala and Asp426 to Glu, due to only two nucleotide changes (T to G at nt
3674 and T to A at nt 4064). Extensive comparative analysis
by BLAST and FASTA indicated that Ser-297 to Ala is shared
by recently isolated CPVs from various parts of the word
(Truyen et al., 2000). In contrast, Glu-426 is a substitution that
is not present in other CPVs or in closely related parvoviruses.
While the change Ser-297 to Ala affects an antigenic
residue close to epitope B over the shoulder region of the
capsid, the change Asp-426 to Glu determines the replacement
of a residue unique to CPV type 2b that is situated in a major
antigenic site, epitope A, over the three-fold spike of the capsid
(Parrish et al., 1991 ; Parrish, 1994 ; Agbandje et al., 1995). After
serial passages in cell culture, there was no apparent modification of either antigenicity or nucleotide composition.
All of the isolates collected in 1998 and 1999 were clearly
identified as type 2b (Table 2). Interestingly, all strains but one
(strain 01\99) had the variation Ser-297 to Ala, suggesting that
this change is common in recent CPVs circulating in Italy.
There were other recurring mutations in some strains, such as
Asp-375 to Asn, Thr-389 to Asn (strains 197\98, 224\98,
244\98 and 111\99) and Ile-418 to Thr (strains 224\98,
244\98 and 197\98).
Both strains 56\00 and 136\00 were collected in 2000, but
on separate occasions and from two distinct veterinary clinics.
This makes it possible to hypothesize that CPVs with
mutations similar to those of strains 56\00 and 136\00 have
been spreading in Italy since the beginning of 2000. An
intriguing question arising from the isolation of strains 56\00
and 136\00 is whether they might represent a newly emerging
variant of CPV. Within a few years of the first recognition of
CPV type 2, the antigenic variants type 2a and 2b replaced the
original CPV type 2 completely. The variants now co-exist,
though with different distributions in different countries. In
Italy, CPV type 2a is prevalent and type 2b is isolated with low
frequency (Sagazio et al., 1998 ; Buonavoglia et al., 2000).
Presumably, these atypical type 2b (56\00-like) CPVs might
currently co-exist in Italy with the other CPV antigenic types.
However, the existence of a new variant should be defined by
a wide distribution throughout the world and by significant
changes in either the biological or antigenic properties of the
virus.
A major point to consider is that all the methods currently
used to distinguish the CPV antigenic types, and routinely
employed in our laboratory, failed to reveal the Glu-426
mutants. The discrimination by MAbs of CPV type 2a from the
variant type 2b is based on only a single MAb (B4A2), which
recognizes type 2a but not type 2b, because the Asn\Asp
change at residue 426 alters the epitope recognized by the
MAbs (Parrish et al., 1991). This means that variations at
DACE
Fig. 1. RFLP analysis of 555for/555rev amplicons. Lanes : M, GeneRuler
100 bp ladder (Fermentas) ; 1, strain 56/00 ; 2, strain 136/00 ; 3, CPV
type 2b ; 4, CPV type 2a.
residue 426, as in strains 56\00 and 136\00, may not be
revealed by this panel of MAbs.
The PCR typing approach was also unable to identify the
variation that occurred in Glu-426 mutants. Type 2b-specific
primers have been designed to take advantage of only two
nucleotide variations, and the variation A G at nucleotide
4062, responsible for the change Asn-426 to Asp, is placed at
the very 3h end of the sense primer (Pereira et al., 2000).
Unfortunately, the Asp Glu change at residue 426 of strains
56\00 and 136\00 is due to a change (T A) in the third
codon position, at nucleotide 4064, and these variants are
erroneously recognized as type 2b by this PCR strategy.
However, since the nucleotide variation at residue 4064 of
strains 56\00 and 136\00 created an MboII restriction site
(GAAGA) unique to these strains, it was possible to distinguish
these mutants from the other antigenic types by simple
digestion of the 555for\555rev amplicon with this enzyme,
which generates two fragments of about 500 and 80 bp
(Fig. 1).
Although the isolation of only two anomalous strains does
not allow the definition of a new antigenic type of CPV, the
occurrence of these natural Glu-426 mutants might indicate a
direction in which evolution is leading CPV. It can not be
excluded that a more detailed examination of recent CPV
strains presenting type 2b may provide evidence for a wider
distribution of 56\00- and 136\00-like CPVs.
We are grateful to Professor C. R. Parrish for his critical comments
and suggestions on the manuscript.
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Received 23 May 2001 ; Accepted 15 August 2001
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