559969

research-article2014

VDIXXX10.1177/1040638714559969Canine parvovirus in MexicoPedroza-Roldan et al.

Brief Communication

Genotyping of Canine parvovirus in

western Mexico

Journal of Veterinary Diagnostic Investigation

2015, Vol. 27(1) 107111
2014 The Author(s)
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DOI: 10.1177/1040638714559969
jvdi.sagepub.com

Csar Pedroza-Roldn,1 Varinia Pez-Magallan, Claudia Charles-Nio,

Darwin Elizondo-Quiroga, Ral Leonel De Cervantes-Mireles,
Mario Alberto Lpez-Amezcua

Abstract. Canine parvovirus (CPV) is one of the most common infectious agents related to high morbidity rates in dogs.
In addition, the virus is associated with severe gastroenteritis, diarrhea, and vomiting, resulting in high death rates, especially
in puppies and nonvaccinated dogs. To date, there are 3 variants of the virus (CPV-2a, CPV-2b, and CPV-2c) circulating
worldwide. In Mexico, reports describing the viral variants circulating in dog populations are lacking. In response to this
deficiency, a total of 41 fecal samples of suspected dogs were collected from October 2013 through April 2014 in the Veterinary
Hospital of the University of Guadalajara in western Mexico. From these, 24 samples resulted positive by polymerase chain
reaction, and the viral variant was determined by restriction fragment length polymorphism. Five positive diagnosed samples
were selected for partial sequencing of the vp2 gene and codon analysis. The results demonstrated that the current dominant
viral variant in Mexico is CPV-2c. The current study describes the genotyping of CPV strains, providing valuable evidence of
the dominant frequency of this virus in a dog population from western Mexico.
Key words: Canine parvovirus; genotyping; Mexico.
Canine parvovirus (CPV) is one of the most common infectious agents causing increased morbidity and mortality in
domestic dogs worldwide. Clinical signs include severe gastroenteritis, vomiting, diarrhea, and dyspnea causing high
mortality in puppies.12 Canine parvovirus belongs to the Parvoviridae family and is a nonenveloped single-stranded DNA
virus with a genome of approximately 5.2 kb. The virus
genome has 2 open reading frames, one encoding the nonstructural proteins NS1 and NS2 and the other encoding the
capsid proteins VP1 (727 residues) and VP2 (584 residues).20
The VP1 and VP2 proteins contain the most important antigenic epitopes, which are targets for neutralizing antibodies.21
It is hypothesized that CPV genotype 2 (CPV-2) emerged
from feline panleukopenia or from a parvovirus in wild carnivores.2 Soon after the appearance of CPV-2 in the mid1980s, new variants emerged and replaced the original
CPV-2 virus; these new antigenic variants were labeled CPV2a and CPV-2b, where CPV-2b differs from CPV-2a by a
single amino acid substitution (Asn to Asp) at position 426 in
the VP2 protein.15 In the year 2000, a new antigenic variant
labeled CPV-2c (Glu at position 426) was identified in Italy.3
Interestingly, within a few years, this new variant partially
displaced CPV-2a and CPV-2b in Italy.13
When last evaluated, the variants CPV-2a, CPV-2b, and
CPV-2c circulate worldwide, and their distributions and
genetic diversities fluctuate among countries.6 For example,
surveillance studies have shown that the 3 CPV-2 variants

are codistributed in Italy,13 Spain,8 and Germany.23 In India,14

Korea,11 and mostly in Asian countries, there is an individual
or codistribution of CPV-2a and CPV-2b. In North America,
specifically in the United States,10 the 3 CPV-2 variants are
co-distributed in contrast with South America where the
most prevalent variant is CPV-2c, as it has been shown in
surveillance studies in Uruguay,18 Brazil,19 and Ecuador.1 In
this regard, surveillance studies of CPV-2 are necessary in
order to understand viral evolution and those factors that
influence dispersion and adaptation of CPV-2 variants in dog
populations. The aim of the current study was to identify and
describe the CPV-2 variants circulating in a dog population
from western Mexico. The results demonstrated a dominant
frequency of CPV-2c in samples collected for the study.
From the Department of Veterinary Medicine, University Center
for Biological and Agricultural Sciences (CUCBA), University of
Guadalajara, Zapopan, Jalisco, Mexico (Pedroza-Roldn, Pez-Magallan,
De Cervantes-Mireles, Lpez-Amezcua); Department of Microbiology and
Pathology, University Center for Health Sciences (CUCS), University of
Guadalajara, Guadalajara, Jalisco, Mexico (Charles-Nio); and Medical
and Pharmaceutical Biotechnology Unit, Center for Research and Applied
Technology in Jalisco (CIATEJ), Guadalajara, Jalisco, Mexico (ElizondoQuiroga).
1

Corresponding Author: Csar Pedroza-Roldn, Departamento de

Medicina Veterinaria, Centro Universitario de Ciencias Biolgicas y
Agropecuarias, Universidad de Guadalajara, Av. Prolongacin Parres Arias
No. 735, Col. Bosques Del Centinela II, CP, 45187, Zapopan, Jalisco,
Mexico. cpedroza46@gmail.com

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Pedroza-Roldn et al.

All clinical samples were collected in the Veterinary Hospital of the University of Guadalajara (Guadalajara, Jalisco,
Mexico), which offers veterinary services to the city of Guadalajara and its metropolitan areas comprised of Zapopan,
Tlaquepaque, Tonala, and Tlajomulco. Samples of all parvovirus suspect cases were collected from October 2013
through April 2014 by rectal swab from vaccinated and
unvaccinated diarrheic dogs. In addition, information such as
date of sampling, age, sex, breed, housing location, and clinical history were recorded. Samples were collected in duplicate and frozen at 20C.
DNA preparation was performed with some modifications, as previously described.18,22 In brief, samples were
thawed at room temperature and homogenized in 1 ml of
saline solution and clarified by centrifugation at 10,000 RCF
for 5 min. Next, samples were diluted 1:10 in distilled water
and heated for 10 min at 98C followed by 5 min at 4C in a
thermal cycler.a Polymerase chain reaction (PCR) amplification of the vp2 gene was performed with oligonucleotidesb
forward (5-GGAAACCAACCATACCAACTCC-3) and
reverse (5-GGATTCCAAGTATGAGAGGC-3) as previously described,17 which generated an amplicon of 1,042 bp
from position 3385 to 4426. The PCR reactions were performed with 3 l of DNA sample, 2.5 l of 10 PCR buffer,
10 pmol/l of each primer, 0.5 l of 10 mmol/l deoxyribonucleotide triphosphate mix, and 0.3 l of Taq DNA polymerasec
(5 U/l) in a final volume of 25 l. Thermal cycler conditions
were initial denaturation at 94C for 5 min, followed by 30
cycles of denaturation at 94C for 30 sec, annealing at 50C
for 1 min, and extension at 72C for 1 min and a final extension at 72C for 10 min. The PCR products were separated in
a 7% polyacrylamide gel electrophoresis and silver-stained.
All PCR-positive samples for the 1,042-bp amplicon were
frozen for further restriction fragment length polymorphism
(RFLP) analysis. Commercial live attenuated vaccined was
used as a positive control, and 5 fecal samples from healthy
dogs were used as negative controls.
The RFLP analysis was performed as previously
described.3,18 In brief, 5 l of the 1,042-bp amplicon was
digested with 2 U of the restriction enzyme MboIIe in the
presence of 1 l of 10 CutSmart buffere and 3 l of distilled
water in a final volume of 10 l. The reactions were incubated at 37C for 1 hr. Samples were subjected to 7% polyacrylamide electrophoresis and silver stain. Cleavage pattern
of the 1,042-bp amplicon was recorded. The 1,042-bp amplicon was subjected to DNA sequencing in both strands using
the oligonucleotides described above. The sequencing process was made in a commercial sequencer.f DNA sequences
were analyzed,g and amino acid sequences were deducedh
and aligned.i Sequences described in the present study were
submitted to GenBank, and accession numbers are described
in Table 2. The CPV-2c reference sequences obtained from
GenBank (Ecuador, KF149984.1, KF149971.1; Uruguay,
KC196109.1, KC196108.1; Italy, FJ222821.1, FJ005249.1)
were used for comparison purposes.

A total of 41 samples were collected from suspected dogs,

and after PCR screening, 24 (58%) were positive for the
1,024-bp amplicon. In a similar way, the live attenuated vaccine showed a same sized band. Nonspecific bands were
observed in the 5 samples obtained from healthy dogs. All 24
samples showed a RFLP pattern associated with the viral
variant CPV-2c, which consisted of 3 bands of 634, 352, and
56 bp. Live attenuated vaccine was used as an internal control and showed a pattern associated with CPV-2a and CPV2b that comprised 2 bands of 634 and 408 bp. This last result
is coherent because the commercial vaccine used in the current study was the Cornell strainbased vaccine. As shown in
Table 1, from the 24 diagnosed dogs with CPV, 16 (67%)
were 6 months or younger, 3 (12%) ranged from 7 to 12
months, and 1 (4%) ranged from 13 to 15 months. Fourteen
dogs (58%) were referred to as never vaccinated, and 7
(29%) dogs received an incomplete vaccine regimen against
CPV; none of the dogs treated in the hospital showed a complete vaccination schedule.
Five random samples were sequenced, and analysis confirmed the sole presence of CPV-2c viral variant with the
characteristic presence of glutamic acid at position 426.
DNA BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) comparison analysis showed a 9899% similarity with most
sequences reported in GenBank; however, the sequences
showed a 99100% similarity at the amino acid level with
most sequences reported worldwide. At the nucleotide level,
CPV-2c sequence comparison against isolates from Ecuador,
Uruguay, and Italy showed several single nucleotide polymorphisms (SNPs). First of all, a nonsynonymous mutation
was identified at position 3442 of the isolate MXGDL/6/11/13, which showed a transition (ATA to ATG) in
the third position of codon 219. This change replaced the
amino acid isoleucine for methionine (Ile to Met), and, to the
authors knowledge, the replacement has not been reported
elsewhere. Additionally, 7 synonymous SNPs were identified along the DNA sequence. For instance, in isolates MXGDL/2/10/13 and MX-GDL/24A/2/14 at position 3583, a
transition (TTT to TTC) in the third position of the codon
was identified. Full descriptions of the SNPs identified in the
present study are described in Table 2.
Since its description in 1970, CPV-2 has had a worldwide
distribution causing high levels of morbidity and mortality in
dog populations. Although dogs from all ages are susceptible
to acquire the infection, puppies or dogs less than 6 months
of age have a higher predisposition for developing the disease.12 As shown in Table 1, dogs diagnosed with the virus
(67%) were not older than 6 months; this is a relatively low
number of infected dogs when compared with reports where
an incidence of 95% was determined.19 However, in the current study, 16% of dogs diagnosed with the virus ranged
from 7 to 15 months. This most likely occurred because the
majority of dogs tested did not have a vaccination program
or were not vaccinated against the virus, therefore increasing
the risk of infection.

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Canine parvovirus in Mexico

109

Table 1. Description of breed, age, vaccination status, and outcome for each Canine parvovirus isolate in the current study.*
Isolate

Breed

MX-GDL/1/10/13
MX-GDL/2/10/13
MX-GDL/3/10/13
MX-GDL/4/10/13
MX-GDL/5/10/13
MX-GDL/6/11/13
MX-GDL/7/11/13
MX-GDL/8/11/13
MX-GDL/19/2/14
MX-GDL/20/2/14
MX-GDL/24A/2/14
MX-GDL/25/2/14
MX-GDL/26/2/14
MX-GDL/28/3/14
MX-GDL/29/3/14
MX-GDL/30/3/14
MX-GDL/31/3/14
MX-GDL/32/3/14
MX-GDL/33/3/14
MX-GDL/34/3/14
MX-GDL/36/3/14
MX-GDL/37/3/14
MX-GDL/38/3/14
MX-GDL/41/4/14

Age

Beagle
Golden Retriever
Belgian Malinois
Basset Hound
NA
St. Bernard
Poodle
Mixed
Mixed
Labrador Retriever
NA
Chihuahua
Miniature Schnauzer
Bull Terrier
Bull Terrier
Chihuahua
American Staffordshire
Terrier
Chihuahua
NA
NA
American Staffordshire
Terrier
Boxer
Mixed
Belgian Malinois

Vaccination status

Outcome

3 months
1 year 5 months
3 months
5 months
NA
4 months
4 months
7 months
5 months
6 months
NA
9 months
3 months
4 months
4 months
7 months
3 months

Incomplete
Incomplete
None
Incomplete
NA
None
None
Incomplete
None
None
NA
None
None
None
None
None
Incomplete

NA
Dead
NA
NA
NA
Dead
Dead
Dead
Dead
Dead
NA
Recovered
Recovered
NA
NA
Dead
NA

5 months
NA
NA
4 months

None
None
NA
Incomplete

Dead
NA
NA
NA

3 months
4 months
3 months

None
None
Incomplete

NA
Dead
Recovered

*NA = not available.

Table 2. Sequence differences of Mexican samples and Canine parvovirus genotype 2c reference strains reported previously.*
Codon and amino acid positions
Isolate

GenBank
Country accession no.

ME28
ME32
M120
M124
56/00
219/08-5
MX-GDL/1/10/13
MX-GDL/2/10/13
MX-GDL/6/11/13
MX-GDL/7/11/13
MX-GDL/24A/2/14

Ecuador KF149984.1 Ile (ATA)

Ecuador KF149971.1 Ile (ATA)
Uruguay KC196109.1 Ile (ATA)
Uruguay KC196108.1 Ile (ATA)
Italy
FJ222821.1 Ile (ATA)
Italy
FJ005249.1 Ile (ATA)
Mexico
KM234975 Ile (ATA)
Mexico
KM234976 Ile (ATA)
Mexico
KM234977 Met (ATG)
Mexico
KM234978 Ile (ATA)
Mexico
KM234979 Ile (ATA)

219

266

270

363

407

425

426

457

Phe (TTT)
Phe (TTT)
Phe (TTT)
Phe (TTT)
Phe (TTT)
Phe (TTT)
Phe (TTT)
Phe (TTC)
Phe (TTC)
Phe (TTT)
Phe (TTC)

Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGT)
Cys (TGC)
Cys (TGT)
Cys (TGT)

Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGG)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)

Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGA)
Gly (GGG)
Gly (GGG)
Gly (GGA)
Gly (GGG)

Thr (ACA)
Thr (ACA)
Thr (ACA)
Thr (ACA)
Thr (ACA)
Thr (ACG)
Thr (ACA)
Thr (ACA)
Thr (ACA)
Thr (ACA)
Thr (ACA)

Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)
Glu (GAA)

Leu (TTA)
Leu (TTA)
Leu (TTA)
Leu (TTA)
Leu (TTA)
Leu (TTA)
Leu (CTA)
Leu (TTA)
Leu (TTA)
Leu (TTA)
Leu (TTA)

*Changes in nucleotide or amino acid are in boldface.

Canine parvovirus 2c has been distributed worldwide and

is cocirculating with the antigenic variants CVP-2a and
CPV-2b. In western Europe, for example, samples analyzed
from Germany, France, and Bulgaria have shown a cocirculation of CPV-2a and 2c, CPV-2b and 2c, and CPV-2a and

2b, respectively.7,9 In contrast, in Hungary and Albania, the

main circulating variant is CPV-2a.4,5 These findings contrast
sharply with reports from South American countries where
the main CPV variant type is 2c, as it has been reported in
Ecuador1 (54%), Brazil19 (78%), and Uruguay17,18 (96%). In

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Pedroza-Roldn et al.

the current study, all samples collected in western Mexico

showed a dominant frequency of the CPV-2c variant as
shown in Table 1. This frequency is only comparable with
studies reported previously in Uruguay, where 24 of 25 samples analyzed were type 2c.18 In Mexico, it might be possible
that CPV-2c has displaced CPV-2a and CPV-2b in the region
and possibly throughout the country. These findings may not
be surprising because viral displacement has been reported
previously in Italy13 and Uruguay.18 However, it is difficult to
asseverate this suggestion because there is a lack of previous
related studies in Mexico with which to compare the viral
dynamics.
Although several nucleotide mutations or SNPs where
identified in samples analyzed in the present study, most of
them were silent with no impact in the codified amino acid.
Interestingly, the isolate MX-GDL/6/11/13 presented a point
mutation (ATA to ATG) that replaced the residue isoleucine
for methionine (Ile to Met) at codon 219. This mutation has
not been reported previously, to the authors knowledge, and
a more in-depth analysis of samples is necessary in order to
determine if this replacement might play a key role in viral
local adaptation (Table 2). Analysis of changes in critical
amino acid positions related with host changes and adaptations,15 such as 297, 300, 305, 323, and 440 of the VP2 protein, of the virus did not show substitutions in reference to
viral variants from Italy, Ecuador, and Uruguay. The presence of alanine (Ala) residue located at position 297 instead
of serine (Ser) residue has been proposed as a key player in
the process of local adaptation and is possibly related with a
viral escape of the immune system in vaccinated animals.16
All analyzed samples herein showed the alanine (Ala) residue at position 297. A similar finding was found in isolates
from South America, and this supports the notion that this
amino acid substitution is increasing in frequency around the
world. In conclusion, the current study demonstrates a dominant occurrence of CPV-2c in the western Mexico region,
and further studies are needed in order to determine if this
CPV-2c dominance is present in other regions of Mexico.
Acknowledgements
The authors wish to thank veterinarians Rebeca Granado, Gabriela
Ramirez, Pedro Hinojosa, Ramon Carlos, Carmina Varela, and students for collecting feces samples used in this study. Also, the
authors thank Michele Brennan for her review of the English language editing.

Sources and manufacturers

a.
b.
c.
d.
e.

Thermal cycler, Thermo Fisher Scientific Inc., Waltham, MA.

Primers, Instituto de Biotecnologia de la UNAM, Cuernavaca,
Mexico.
PCR reagents, Vivantis Technologies Sdn. Bhd., Subang Jaya,
Malaysia.
Parvomune, Holland Animal Health, Cuernavaca, Mexico.
MboII enzyme, 10 CutSmart buffer; New England Biolabs
Inc., Ipswich, MA.

f.
g.
h.
i.

ABI PRISM 3100, Applied Biosystems, Foster City, CA.

FinchTV version 1.5, Geospiza Inc., Seattle, WA.
Serial cloner 2.6.1, SerialBasic Software, France.
BioEdit Biological Sequence Alignment Editor, Ibis Biosciences, Carlsbad, CA.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and publication of this article.

Funding
This work was supported in part by the Department of Veterinary
Medicine under the project P3e-2014 with the reference number
220731 approved to CPR.

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