DOI: 10.7589/2016-08-194 Journal of Wildlife Diseases, 53(4), 2017, pp.

824–831
Ó Wildlife Disease Association 2017

GENETIC CHARACTERIZATION OF CANINE PARVOVIRUS IN

SYMPATRIC FREE-RANGING WILD CARNIVORES IN PORTUGAL
Carla Miranda,1,2 Nuno Santos,2 Colin Parrish,3 and Gertrude Thompson1,2,4
1
Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal
2
Rede de Investigação em Biodiversidade e Biologia Evolutiva (CIBIO/InBIO), Laboratório Associado, Universidade do
Porto, 4485-661 Vairão, Portugal
3
Baker Institute for Animal Health, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell
University, 235 Hungerford Hill Road, Ithaca, New York 14853, USA
4
Corresponding author (email: gat1@mail.icav.up.pt)

ABSTRACT: Since its emergence in the 1970s, canine parvovirus (CPV) has been reported in domestic
and nondomestic carnivores worldwide with severe implications on their health and survival. Here, we
aim to better understand CPV circulation in multihost-pathogens systems by characterizing CPV DNA
or viruses in 227 free-ranging wild carnivores of 12 species from Portugal. Collected samples during
1995–2011 were analyzed by PCR and sequence analysis. The canine parvovirus DNA was detected in 4
(2%) animals of two species, namely in wolves (Canis lupus; 3/63, 5%, 95% confidence interval¼1.6–
3.15) and in a stone marten (Martes foina; 1/36, 3%, 95% confidence interval¼0.5–14.2). Viruses in two
wolves had VP2 residue 426 as aspartic acid (so-called CPV-2b) and the third had VP2 residue 426 as
asparagine (CPV-2a), while the virus in the stone marten uniquely had VP2 residue 426 as glutamic acid
(CPV-2c). The comparative analysis of the full-length VP2 gene of our isolates showed other
nonsynonymous mutations. The phylogenetic analysis demonstrated that the sequences from wolves
clustered together, showing a close relationship with European domestic dogs (Canis lupus familiaris)
and wolf strains while the viral sequence from the stone marten grouped with other viruses contained
the glutamic acid VP2 426 along with raccoon (Procyon lotor), bobcat (Lynx rufus), and domestic dog
strains. This study confirmed that wild carnivores in Portugal are infected by CPV variants, strongly
suggesting viral transmission between the wild and domestic populations and suggesting a need for a
better understanding of the epidemiology of the disease and its management in wild populations.
Key words: Canine parvovirus, carnivores, Parvoviridae, Portugal, stone marten, wolf.

INTRODUCTION Although CPV-2 has a single-stranded DNA

Efficient transmission of viruses in new host
species is a major obstacle to their successful
cross-species transmission and emergence.
Emergent viruses generally appear maladapt-
ed to their new host species and therefore
cause only short transmission chains. Evolu-
tion may help viruses surmount species
barriers, principally by affecting virus-host
interactions (Kuiken et al. 2006). Hence, the
process of viral emergence may require
multistage adaptations to a new host, an idea
supported by the observation that RNA
viruses (and some single-stranded DNA vi-
ruses), which often exhibit evolutionary plas-
ticity, may arise as emerging diseases
(Woolhouse et al. 2001).
Canine parvovirus type 2 (CPV-2) repre-
sents an example of successful cross-species
viral transmission (Hoelzer et al. 2008).
824
genome and uses the cellular replication
machinery, estimates of nucleotide substitu-
tion rates are more comparable to those
observed in RNA viruses than in other DNA
viruses (Shackelton et al. 2005). The CPV-2
arose in the mid-1970s as a host range variant
of an endemic parvovirus of cats, mink, or
related hosts within the order Carnivora
(Hoelzer et al. 2008), but canids and some
related carnivores had previously resisted
infection by those viruses (Parrish 1990).
The emergence of CPV-2 in dogs was
associated with the virus acquiring the ability
to bind the canine transferrin receptor type-1
through changes in its capsid protein. Differ-
ences on the top and the side of the threefold
spike of the capsid surface controlled specific
transferrin receptor type-1 binding and the
efficiency of binding to feline and canine cells
(Hueffer et al. 2003).
 MIRANDA ET AL.—CPV CHARACTERIZATION IN WILD CARNIVORES
The CPV-2 that emerged in 1978 was
replaced in 1980 by a genetic and antigenic
variant, designated CPV-2a, due to five or six
amino acid changes located in the VP2 gene
(Parrish et al. 1988). Additional variants have
since arisen that have various changes in their
capsids, including the substitution of aspara-
gine (Asn) for aspartic acid (Asp, previously
called CPV-2b) or glutamic acid (Glu, previ-
ously called CPV-2c) at VP2 residue 426
(Parrish et al. 1988, 1991; Buonavoglia et al.
2001). The CPV-2a and its derivatives re-
gained the ability to infect felids (Truyen et al.
1996); they are globally distributed, and infect
a variety of domestic and wild hosts (Barker et
al. 1983; Parrish 1990; Allison et al. 2012). A
recent study showed that the cross-species
transfer of viruses between carnivores and
different hosts is determined in particular by
changes of VP2 residue 300, as well as some
other changes in the capsid (Allison et al.
2016).
The importance of infectious diseases in
carnivore conservation and their role in
population dynamics and declines have al-
ready been described for several wildlife
species. Hence, limiting contact between
domesticated hosts and wildlife could reduce
risks to wildlife populations (Pedersen et al.
2007). Infection with CPV has already been
shown to occur in Canidae, Felidae, Procyo-
nidae, and Mustelidae families of the order
Carnivora (Barker and Parrish 2001; Steinel et
al. 2001). A variety of information is available
on the genetic characterization of CPV in
carnivores (Steinel et al. 2000; Allison et al.
2012, 2013), and several studies have reported
the detection of CPV antibodies in wild
carnivores (Sobrino et al. 2008; Woodroffe et
al. 2012; Watts and Benson 2016).
Infectious viral diseases can serve as
important morbidity and mortality factors
and may influence wolf (Canis lupus) popu-
lation demography through direct mortality
and diminished recruitment (Mech et al.
2008). Canine parvovirus was a significant
factor retarding recolonization and population
increase for wolves in Minnesota (Mech et al.
2008). The Iberian wolf (Canis lupus signatus)
is classified as Endangered in Portugal (Pi-
825
menta et al. 2005) and one conservation goal
is to diminish their mortality, including that
caused by infectious diseases. In fact, wolves
are susceptible to a large spectrum of canine
pathogens commonly found in domestic dogs,
including CPV (Murray et al. 1999; Oleaga et
al. 2015; Millán et al. 2016). A serologic study
(Santos et al. 2009) suggested the exposure of
wolves, red foxes (Vulpes vulpes), wildcats
(Felis silvestris), common genets (Genetta
genetta), and stone martens (Martes foina) to
CPV infection in Portugal and, more recently,
Duarte et al. (2013) typified a stone marten
virus as CPV-2 with the substitution of VP2
residue 426 to Asp. Here, we tested for the
presence of CPV in samples from 227 free-
ranging wild carnivores (12 species) from
Portugal.
MATERIALS AND METHODS
Samples of 227 free-ranging wild carnivores
from several species of the order Carnivora were
analyzed in this study (Table 1); they were
collected in Portugal from 1995 to 2011 and
stored at 20 C in the Banco de Tecidos de
Vertebrados Selvagens and Sistema de Monitor-
ização de Lobos Mortos, Portugal. These included
tissues of animals found dead by road kill (n¼111),
loop (n¼11), shooting (n¼10), poisoning (n¼3),
infectious disease (n¼7), and other causes (n¼40).
It was not possible to determine the cause of
death in 45 cases. No animals were sacrificed for
the purposes of this specific study, and the
authors were not responsible for the death of
any animals. During standard necropsy proce-
dures, cadavers were grossly classified as fresh
(n¼100), medium (n¼53), and heavily autolyzed
(n¼57) while no information was available for 17
cadavers.
The study area included continental Portugal
(Fig. 1). We determined sex for the majority of
the animals and age was determined according to
tooth eruption and wear (Table 1). The tissue
samples analyzed in this study included liver
(n¼116), spleen (n¼70), lung (n¼19), and heart
(n¼22).
Approximately 30 mg of tissue sample were
used for viral DNA preparation, as previously
described by Desario et al. (2005). For CPV
detection, the primers amplifying the entire VP2
gene (1,755 nucleotides) were used, as previously
described by Miranda et al. 2016. The primer
sequences are available from the authors by
request. The PCR products were sequenced at a
826 JOURNAL OF WILDLIFE DISEASES, VOL. 53, NO. 4, OCTOBER 2017

TABLE 1. Families and species of free-ranging wild carnivores tested for the detection of canine parvovirus,
collected in continental Portugal from 1995 to 2011.

Age Sex
a
Family, Species Common name Juvenile Adult ND Female Male NDa Total tested

Canidae
Canis lupus Gray wolf 22 41 31 32 63
Vulpes vulpes Red fox 4 17 4 11 13 1 25
Felidae
Felis silvestris Wild cat 3 21 12 10 24 2 36
Viverridae
Genetta genetta Common genet 5 21 6 17 14 1 32
Herpestes ichneumonn Mongoose 1 1 1
Mustelidae
Martes foina Stone marten 3 26 7 11 20 5 36
Martes martes Pine marten 5 1 4 5
Lutra lutra Eurasian otter 10 5 5 10
Meles meles Eurasian badger 1 6 3 4 7
Mustela putorius European polecat 5 2 1 6 7
Mustela vison American mink 2 1 1 2 3
Mustela nivalis Weasel 1 1 2
a
ND ¼ no data.

commercial laboratory after they had been
purified using the NZY Gel pure kit (NZYTech
Genes and Enzymes, Lisbon, Portugal). The
sequences were assembled, edited, and aligned
with reference strains obtained from the GenBank
database using the Geneious R6 software (Bio-
matters Ltd., Auckland, New Zealand).
A maximum likelihood phylogenetic tree of the
sequences was obtained using MEGA6 (Tamura
et al. 2013). The phylogenetic tree was construct-

FIGURE 1. Geographic distribution of the free-ranging wild carnivores collected in continental Portugal from
1995 to 2011. The animal species are represented in the map by symbols, such as those identified as canine
parvovirus (CPV)-positive carnivores.
 MIRANDA ET AL.—CPV CHARACTERIZATION IN WILD CARNIVORES
ed using the full-length sequences of VP2 region
deposited in GenBank and estimated using
bootstrap values from 1,000 replicates.
RESULTS
Of the 227 samples from wild carnivores,
CPV DNA was detected in four (2%, 95%
confidence interval [CI]¼0.7–4.4) animals,
specifically in wolves (3/63, 5%, 95%
CI¼1.6–3.2) and a stone marten (1/36, 3%,
95% CI¼0.5–14.2). Positive wolves were
collected in Northern Portugal (Fig. 1) in
1996 (41846 0 49 00 N , 6840 0 39 00 W ), 2002
( 4 1 81 7 0 3 9 00 N , 7 85 0 0 4 8 00 W ) , a n d 2 0 0 5
(41844 0 17 00 N, 8812 0 3 00 W). These comprised
one male (juvenile) and two females (one
juvenile and one adult). The juvenile female
was killed by an illegal snare while cause of
death was undetermined for the other two
wolves. The stone marten was an adult female
that was found dead as a road kill in Central
Portugal (40859 0 55 00 N, 6856 0 19 00 W) in 2008.
According to the sequence at VP2 residue
426, two wolves were infected with CPV-2b
(W33/PT/96 and W52/PT/05) and one with
CPV-2a (W50/PT/02). The stone marten virus
was classified as CPV-2c (Sm14/PT/08). Am-
plification of the full VP2 gene for sequencing
was only successful with specific DNA from
samples from two of the three wolves that
tested CPV DNA-positive. The full-length
VP2 sequences were deposited in GenBank
(accession nos. KU662349–KU662351). The
comparative analysis of these showed non-
synonymous mutations in all isolates. Amino
acids occupying positions 87 (leucine), 300
(glycine; Gly), and 305 (tyrosine), which are
characteristic of CPV-2a strains, were found
in wolves and the stone marten; however, the
amino acid 101 changed (from isoleucine to
threonine) in a wolf (W33/PT/96). In addition
to these, there were new nonsynonymous
mutations located at residue 115 (Asn to
isoleucine), 245 (threonine to Asn), and 247
(Glu to alanine) in wolf isolates (W52/PT/05).
The stone marten (Sm14/PT/08) infected by
the CPV isolate with the 426Glu change
revealed additional changes in residues 99
(Asp acid to alanine), 115 (Asn to histidine),
827
156 (serine to phenylalanine), and 233 (thre-
onine to phenylalanine).
Phylogenetic analysis of the complete VP2
nucleotide sequences isolated from domestic
and wild animals in Portugal and worldwide
(Fig. 2), as well as a feline panleukopenia
virus sequence as an outgroup, showed that
both obtained sequences from wolves clus-
tered in same group as European dogs and
Italian wolf strains. The stone marten se-
quence grouped alone within the CPV-2a
426Glu cluster, although showing 99.4%
identify with another strain from the same
species (GenBank accession no. JX411926;
Fig. 2).
DISCUSSION
Many pathogens can infect more than one
species of host (Woolhouse et al. 2001), and
this is seen for CPV, which has become a
common virus in many carnivores besides the
domestic dog (Truyen et al. 1996). Parvovirus
virions are extremely stable and can remain
infectious in the environment for many
months, which facilitates transmission by the
fecal-oral route among susceptible species
(Truyen et al. 1998). The CPV variants have
been shown to circulate widely among wildlife
(Steinel et al. 2000; Allison et al. 2014). Large
carnivores are of vital importance to the
stability and integrity of most ecosystems,
but declines in free-ranging populations have
highlighted the potentially devastating effect
of infectious diseases on their conservation
(Murray et al. 1999). Pedersen et al. (2007)
suggested that the close evolutionary similar-
ity between wild and domesticated animals
may be a key factor associated with infectious,
pathogen-mediated declines of wildlife; thus,
efforts to limit contact between domestic
hosts and wildlife could reduce spillover and
extinction risk.
The present study summarizes the molec-
ular survey of CPV in wild carnivores in
Portugal during 17 y (1995–2011). We
identified the presence of CPV variants in
wolves and a stone marten. These results are
in agreement with a recent study in which
828 JOURNAL OF WILDLIFE DISEASES, VOL. 53, NO. 4, OCTOBER 2017

FIGURE 2. Phylogenetic analysis of the VP2 complete nucleotide sequences of canine parvovirus from two
wolves and one stone marten obtained in this study (indicated by a solid circle), other wild carnivore parvovirus
(B¼bobcat, Lynx rufus; C¼coyote, Canis latrans; F¼fisher, Martes pennanti; Lc¼leopard cat, Felis bengalensis;
 MIRANDA ET AL.—CPV CHARACTERIZATION IN WILD CARNIVORES
we demonstrated that variants of CPV
isolated from domestic dogs in Portugal were
predominantly the 426Glu variant, followed
by the 426Asp and 426Asn viruses (Miranda
et al. 2016), strongly suggesting viral trans-
mission between the wild and domestic
populations. In addition, our findings also
agree those of Santos et al. (2009) and
Duarte et al. (2013), which have shown the
presence of CPV antibodies in wolves, red
foxes, wildcats, common genets, and stone
martens and of CPV DNA in a stone marten,
respectively.
In Bulgaria, two out of 57 samples (approx-
imately 4%) from wild carnivores were
parvovirus DNA positive by PCR, with a wolf
and a red fox both being infected by 426Asn
strains (Filipov et al. 2014). Virus from wolves
has been characterized as 426Glu variants in
Italy by Battilani et al. (2001) and 426Asp and
Glu in the US by Allison et al. (2013, 2014).
Thus, our findings are in agreement with the
CPV variants reported in European wolves
(Filipov et al. 2014). Our phylogenetic anal-
yses show a close genetic similarity of the CPV
isolates from Portuguese wolves with the
Italian wolf and domestic dogs strains from
Germany and the US. A similarity between
viruses of wolves and domestic dogs suggests
that the wolf virus may have been spillover
from infected domestic species due to direct
or indirect contact. On the other hand, only
two isolates from stone martens revealed the
presence of CPV in this species, one collected
in Portugal that contained 426Asp (Duarte et
al. 2013) and another described (Steinel et al.
2001) as a CPV-2a (426Asn) strain, whose
nucleotide sequence is not available in the
GenBank database. Our findings present the
first report of the molecular detection of CPV
with 426Glu in a stone marten, with further
characterization of the nucleotide sequence of
VP2 gene.
Mpc¼masked palm civet, Paguma larvata; P¼puma, Puma concolor; R¼raccoon, Procyon lotor; Rf¼red fox,
Vulpes vulpes; Ro¼river otter, Lontra canadensis; Sm¼stone marten, Martes foina; W¼wolf, Canis lupus), and
from domestic dog sequences available in the GenBank database. Feline panleukopenia virus (Cat, Felis catus)
was used as an outgroup. The percentage of bootstrap support (.70%) is indicated above the branches.
829
We could also show the presence of several
additional mutations. The change of residue
297 (alanine) in the stone marten is commonly
seen (Duarte et al. 2013), while VP2 position
300 is present as several different variations in
different carnivore hosts, including Asp, Gly,
alanine, serine, and valine (Qin et al. 2007;
Chen et al. 2011; Allison et al. 2016). The
ability of raccoon viruses to infect dog cells
has been associated with a single mutation of
VP2 residue 300, from Asp to Gly—the same
change that has been found in recent isolates
of CPV from dogs which have a residue
300Gly described in domestic cats, coyote,
and gray wolf (Allison et al. 2016) and in the
stone marten strains reported here and by
Duarte et al. (2013). These mutations dem-
onstrate that this virus continues to evolve and
can readapt within its host species.
The effects of CPV in Portuguese wildlife
populations are currently unknown. The low
prevalence results (5%) found in this recent
work suggest that CPV infection is probably
not a major factor limiting the wolf popula-
tion. Pathogens such as CPV may not
significantly affect morbidity or mortality
alone within wildlife populations, but the
synergistic effects of concurrent infections
(for example, increased physiologic stress,
immunosuppression, and other viruses) may
have important consequences (Watts and
Benson 2016). Our study provides a useful
contribution to the understanding of the
carnivore parvovirus evolution and epidemi-
ology, thus helping to address strategies of
disease control in wildlife.
ACKNOWLEDGMENTS
This work was supported in part by the
Foundation for Science and Technology (FCT)
Portugal grant SFRH/BD/76291/2011 to C.M.,
and others such as the Human Potential Oper-
ational Programme (POPH), and the European
Union. Thanks are due to Banco de Tecidos de
Vertebrados Selvagens (BTVS/ICNF) and Siste-
830 JOURNAL OF WILDLIFE DISEASES, VOL. 53, NO. 4, OCTOBER 2017
ma de Monitorização de Lobos Mortos (SMLM/
ICNF) for providing access to the biologic
samples. The authors declared no potential
conflicts of interest with respect to the research,
authorship, or publication of this article.
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Submitted for publication 21 August 2016.
Accepted 1 December 2016.