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hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks
Martella Vito, Elia Gabriella, Lucente Maria Stella, Decaro Nicola, Lorusso Eleonora, Banyai Krisztian, Blixenkrone-Møller Merete, Lan Nguyen Thi,
Yamaguchi Ryoji, Cirone Francesco, et al.
To cite this version:
Martella Vito, Elia Gabriella, Lucente Maria Stella, Decaro Nicola, Lorusso Eleonora, et al.. Geno- typing Canine Distemper Virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks. Veterinary Microbiology, Elsevier, 2007, 122 (1-2), pp.32.
�10.1016/j.vetmic.2007.01.005�. �hal-00532193�
Title: Genotyping Canine Distemper Virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks
Authors: Martella Vito, Elia Gabriella, Lucente Maria Stella, Decaro Nicola, Lorusso Eleonora, Banyai Krisztian,
Blixenkrone-Møller Merete, Lan Nguyen Thi, Yamaguchi Ryoji, Cirone Francesco, Carmichael Leland Eugene, Buonavoglia Canio
PII: S0378-1135(07)00013-2
DOI: doi:10.1016/j.vetmic.2007.01.005
Reference: VETMIC 3559
To appear in: VETMIC Received date: 13-10-2006 Revised date: 22-12-2006 Accepted date: 2-1-2007
Please cite this article as: Vito, M., Gabriella, E., Stella, L.M., Nicola, D., Eleonora, L., Krisztian, B., Merete, B.-M., Thi, L.N., Ryoji, Y., Francesco, C., Eugene, C.L., Canio, B., Genotyping Canine Distemper Virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks,Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.01.005
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Accepted Manuscript
1
Genotyping Canine Distemper Virus (CDV) by a hemi-nested multiplex
1
PCR provides a rapid approach for investigation of CDV outbreaks
2
3
Martella Vito*,1 Elia Gabriella,1 Lucente Maria Stella,1 Decaro Nicola, 1 Lorusso
4
Eleonora,1 Banyai Krisztian,2 Blixenkrone-Møller Merete,3 Lan Nguyen Thi, 4 Yamaguchi
5
Ryoji, 4 Cirone Francesco,1 Carmichael Leland Eugene,5 Buonavoglia Canio.1
6 7
1 Department of Animal Health and Well-being, University of Bari, Valenzano, Bari, Italy 8
2 Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pecs, Hungary 9
3 Laboratory of Virology, Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural 10
University, Stigbojlen 7, 1870 Frederiksberg C, Copenhagen, Denmark.
11
4 Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan, 889-2192 12
5 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 13
14853, US 14
15
Running title: genotyping of vaccine and field CDV strains.
16 17
*Corresponding author:
18
Vito Martella - Dipartimento di Sanità e Benessere Animale, Facoltà di Medicina Veterinaria
19
di Bari, S.p. per Casamassima km 3, 70010 Valenzano, Bari, Italia
20
Tel: 39 080 4679805
21
Fax: 39 080 4679843
22
E-mail: v.martella@veterinaria.uniba.it
23
Accepted Manuscript
ABSTRACT
1
CDV is a highly contagious viral pathogen causing a lethal systemic disease in dogs
2
and other carnivores. Several lineages or genotypes of CDV exist that are variously distributed
3
throughout several continents. Legal or uncontrolled trading of animals may modify the
4
epidemiology of CDV, introducing novel strains in CDV-naïve areas or accounting for the
5
resurgence of CDV in areas where vaccine prophylaxis was effective and successful to control
6
the disease. A hemi-nested PCR system was developed to genotype strains of the major CDV
7
lineages, America-1, Europe, Asia-1, Asia-2 and Arctic. The assay was tested using a
8
collection of 27 laboratory and vaccine strains and of 36 field CDV strains. Distinct lineages
9
could be differentiated by specific primers targeted to the H gene. The method could be useful
10
for molecular epidemiological studies of CDV, providing a tool for large-scale studies, and for
11
the diagnosis of vaccine-related disease.
12 13
Keywords: Canine distemper virus, characterization, protein H, RT-PCR
14 15 16 17 18 19 20 21 22 23
Accepted Manuscript
1. Introduction
1
Canine distemper virus (CDV) belongs to genus Morbillivirus in the Paramyxoviridae
2
family. The virus possesses a single-stranded negative RNA that encodes for a single envelope-
3
associated protein (M), two glycoproteins, [hemagglutinin/attachment protein (H) and fusion
4
protein (F)], two transcriptase-associated proteins (the phosphoprotein P and the large protein
5
L), and the nucleo-capsid protein (N), that encapsidates the viral RNA (van Regenmortel et al.,
6
2000).
7 8
CD is an acute to subacute contagious systemic disease with high mortality rates in
9
dogs and wild carnivores. The disease is characterized by pyrexia, nasal and ocular discharge,
10
gastrointestinal and respiratory symptoms and nervous signs (Appel, 1987). The infection and
11
the disease are not age-restricted and may be prevented by passive or active immunization
12
(Appel, 1987).
13 14
The introduction of modified live (ML) CDV vaccines in the 1950s and their extensive
15
use has greatly helped to keep the disease under control (Appel, 1987; Appel and Summers,
16
1995; Greene and Appel, 1998). Notwithstanding, the incidence of CDV-related disease in
17
canine population throughout the world seems to have increased in the last decades and several
18
episodes of CDV disease in vaccinated animals have been reported (Blixenkrone-Møller et al.,
19
1992; Decaro et al., 2004; Frolich et al., 2000; Gemma et al., 1996a and 1996b; Kai et al.,
20
1993; Patronek et al., 1995; Scagliarini et al., 2003).
21 22 23
Accepted Manuscript
Molecular epidemiology is useful to trace the origin of CDV strains and to investigate
1
the dynamics of circulation of the various strains in susceptible animals. Unfortunately, the
2
lack of common standards and protocols in epidemiologic studies of CDV does not allow
3
precise evaluation of the epidemiologic features of this important pathogen, since previous
4
studies have targeted different genome regions. Sequence analysis of CDV strains identified in
5
different geographical settings and from various animal species has revealed that the H
6
gene/glycoprotein of CDV undergoes a genetic/antigenic drift, according to geographic
7
patterns related to the geographic location of the circulating strains. In addition, the H gene has
8
been shown to be a reliable target to reconstruct and investigate the genetic relationships
9
among CDV strains. The majority of CDV field strains cluster into six major genetic lineages,
10
designated America-1 and –2, Asia-1 and –2, European and Arctic (Bolt et al., 1997; Carpenter
11
et al., 1998; Haas et al., 1997; Harder et al., 1996; Hashimoto et al., 2001; Iwatsuki et al., 1997;
12
Lan et al., 2005b and 2006; Lednicky et al., 2004; Martella et al., 2002 and 2006; Mochizuki
13
et al., 1999; Pardo et al., 2005) (Figure 1). Current CDV ML vaccines have been produced
14
using CDV isolates within the America-1 lineage. The Snyder Hill strain was isolated in Ithaca,
15
N.Y., USA in the 1950's from the brain of a dog and passaged in vivo in dogs before being
16
adapted to cell growth in NL-DKC cells (Brown et al., 1972). The Onderstepoort strain, used
17
worldwide as an attenuated live vaccine, dates back to a disease outbreak among North
18
American ranched foxes in the 1930’s (Haig, 1956). America-1 CDVs have not been detected
19
over the last 5 decades and it is not known whether they are still circulating in the field.
20
Recently, vaccine-like CVDs have been detected from outbreaks in wildlife animals but they
21
have been reported only occasionally in dogs (Harder and Osterhaus, 1997; Lednicky et al.,
22
2004). In addition, the detection of Arctic CDV strains, formerly believed to circulate
23
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exclusively in the Arctic ecosystem, has been reported in United States (Pardo et al., 2005) and
1
Europe (Martella et al., 2006) raising questions on the origin of these unusual strains.
2
Uncontrolled trading of animals has been conjectured as one reason for the observed changes
3
in CDV epidemiology as a consequence of the introduction of novel strains into CDV-naïve
4
areas or into countries where CDV spread had been effectively controlled by vaccination.
5 6
Based on the genetic heterogeneity in the H gene, an RT-PCR genotyping assay was
7
developed that can differentiate CDV strains of vaccine origin from field strains as well as
8
predict the genotype of field strains. The system described here was validated in a multicentric
9
study involving three laboratories in Japan and Europe. Tests were done on strains representing
10
all CDV genotypes identified thus far that are considered epidemiologically relevant.
11 12
2. Materials and methods
13
2.1 Viruses and clinical specimens
14
Thirty-eight CDV-positive cases were identified in our laboratories from 2002 through
15
April 2006 by screening animals affected with CDV-associated symptoms, e.g. neurological
16
signs, enteritis, respiratory distress, nasal and ocular discharge, fever. Samples were submitted
17
by animal hospitals in several regions of Central and Southern Italy and CDV RNA was
18
detected by reverse transcription (RT)-PCR, using primer pair P2-P7 that amplifies a 478-bp-
19
long fragment of the N gene (Shin et al., 1995) and by a real-time PCR assay (Elia et al., 2006).
20 21
The canine strains DK91B and DK91C and the mink strain DK86, isolated in Denmark
22
(Blixenkrone-Møller et al., 1992; Blixenkrone-Møller et al., 1993) were used as prototypes of
23
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the European CDV genotype. Strain GR88 was detected from a sledge dog in remote Inuit
1
settlement in Northern Greenland (Blixenkrone-Møller et al., 1992) and was used as prototype
2
of the Arctic genotype. Italian strains 179/04 and 48/04 were detected in 2004 and 2005 in
3
Rome and Messina, respectively, and also were used as reference strains of the Arctic lineage
4
(Martella et al., 2006). Strains KDK-1, S124C, Ac96I and P94S (Japan) were used as reference
5
strains of lineage Asia-1, and strains 007Lm, 009L and 011C (Japan) as reference strains of
6
lineage Asia-2 (Lan et al., 2005a and 2005b and 2006; Mochizuki et al., 1999). The vaccine
7
strains Onderstepoort (five different vaccine products, A to E), Snyder Hill and “Distemperoid”
8
were obtained from vaccines obtained from the market. In addition, three strains of the
9
reference Onderstepoort virus and a Snyder Hill strain, maintained in our laboratories, were
10
included in the analysis as prototypes of the genotype America-1. The strain Javelina/US89
11
(Appel et al., 1991), Dog/US89 (Blixenkrone-Møller et al., 1992) and Leopard/US91 (Appel et
12
al., 1994) were used as prototypes of the genotype America-2. The vaccine and reference
13
strains used in this study are listed in Table 1.
14 15
2.2 Genotyping strategy and primer selection
16
Primers were synthesized in Tib Molbiol laboratories (Genoa, Italy). The typing
17
primers were selected taking advantage of nucleotide polymorphisms conserved within strains
18
of each lineage, but divergent among strains of the various lineages. Three specific typing
19
primers (minus-sense) were prepared for gene H sequences of strains of lineage Europe (primer
20
200, prototype strain DK91A), lineage America-1 (primer 201b, prototype strain
21
Onderstepoort), and lineage Arctic-like (primer 202b, prototype strain GR88). An additional
22
two specific typing primers (minus-sense) were prepared for gene H sequences of strains of
23
Accepted Manuscript
lineage Asia-1 (primer 199b, prototype strain KDK-1) and Asia-2 (primer 203b, prototype
1
strain HM-6). Due to the heterogeneity of strains and to lack of conserved nucleotide
2
polymorphism, it was not possible to design primers specific for the lineage America-2. One
3
pair of primers, 204-232b, made against the consensus sequence of 55 gene H sequences
4
available in the databases, was chosen for the first RT-PCR amplification, in regions that are
5
highly conserved among CDV strains and include all the lineage-specific polymorphisms
6
useful for the genotyping strategy. The nucleotide (nt) positions and sequences of the primers
7
are shown in Table 2. The genotyping strategy is outlined in Figure 2.
8 9
Reverse transcription and PCR amplification of the H gene of CDV were achieved in a
10
single step procedure. Total RNA was obtained from 25 mg samples of tissue homogenates or
11
swab washes. The RNA was extracted using the RNeasy Kit (Qiagen, Gmbh, Germany)
12
according to the manufacturer's instructions. The RNA was eluted in 50 µl of RNase-free
13
water. To obtain a PCR product of the H gene, 0.5 µl each of primers 231 and 232 (50 pmol/µl)
14
was added to a total of 24 µl of the reaction mixture containing 0.2 mM of each dNTP, 1.2 mM
15
MgSO4 and 0.5 µl of a mix of SuperScript II H- Reverse Transcriptase and Platinum Taq HiFi
16
(Invitrogen - Life Technologies, Milan, Italy). The RNA was reverse transcribed and
17
immediately subjected to PCR amplification in a single-step protocol, using SuperScript One-
18
Step RT-PCR kit (Invitrogen - Life Technologies, Milan, Italy). Reverse transcription was
19
carried out at 48°C for 60 min, followed by denaturation of the reverse transcriptase at 95°C
20
for 2 min. Amplification was conducted by a temperature cycling protocol consisting of 40
21
cycles of 30 s for denaturation at 94°C, 30 s of primer annealing at 50°C, and 1.5 min of
22
extension at 68°C, followed by 10 min of final extension at 68°C. With this RT-PCR a
23
Accepted Manuscript
fragment of 1160 bp of the gene H of CDV was amplified. The cDNA was diluted 1: 200 in
1
distilled water and used as template for the second-round PCR. In this PCR, portions of the
2
1160-bp cDNA were amplified using a cocktail of primers that includes the plus-sense primer
3
204 and the minus-sense lineage-specific primers 199, 200, 201, 202, 203b. Selection of
4
specific primers that gave products of different size permitted identification of the gene H
5
genetic lineage group by agarose gel analysis. The use of a cocktail of primers has the added
6
advantage of identifying in one PCR the lineage of any CDV isolate. For the typing reaction
7
(second-round PCR), 1 µl of the diluted cDNA was added to a total of 49 µl of reaction
8
mixture consisting of 0.25 µl of AmpliTaq Gold (1.25 U) (Applera, Milan, Italy), 5 µl of 10×
9
PCR buffer, 2 µl of 2.5 mM dNTP mixture, 35.25 µl of distilled H2O, 0.25 µl of each primer
10
(50 pM). After 10 min at 94°C for activation of DNA polymerase, amplification was conducted
11
by a temperature cycling protocol consisting of 25 cycles of 1 min of denaturation at 94°C, 1
12
min of primer annealing at 50°C, and 1 min of extension at 72°C, followed by 10 min of final
13
extension phase at 72°C.
14 15
2.3 Sequence analysis of the full-length gene H
16
To confirm the results of the genotyping strategy, eight CDV strains were selected to
17
obtain sequence information on the H gene. Reverse transcription and PCR amplification of the
18
full-length H gene of CDV were achieved with primers RH-3 and RH-4 as previously
19
described, with minor modifications (Mochizuki et al., 1999). The RH3-RH4 PCR products
20
were purified with Ultrafree-DA Columns (Amicon, Millipore). The DNA was then used as
21
template for direct sequencing (Genome Express, Meylan France). The DNA was sequenced
22
using the conserved primers RH3 and RH4 and specific primers designed according to an
23
Accepted Manuscript
overlapping strategy. The sequences were then assembled using Bioedit software package
1
version 2.1 (Hall, 1999).
2 3
3. Results
4
3.1 Gene H typing and sequence analysis of the H gene
5
Analysis of seven vaccine CDV strains (five Onderstepoort-based vaccines, one Snyder
6
Hill-based vaccine and one ‘Distemperoid’ vaccine), of three Onderstepoort strains and of one
7
Snyder Hill strain available in our laboratory, of 16 reference CDV strains from our laboratory
8
collection and of 36 field strains was carried out by two PCR amplifications. In the first PCR,
9
the primers 204 and 232b yielded intense DNA products of the predicted size (1160 bp) for all
10
63/63 strains tested (Figure 3). Some nonspecific bands were observed sporadically. The nature
11
of nonspecific but discrete products was not characterized but it probably resulted from
12
mispriming events during amplification of the nucleic acids. Those products did not interfere
13
with gene H typing.
14 15
RNA concentrations were determined by using a real time RT-PCR specific for CDV
16
(Elia et al., 2006). The RT-PCR sensitivity (primers 204/232b) was evaluated by using samples
17
containing known amounts of viral RNA. Samples containing 107 to 102 copies of viral RNA,
18
negative tissue samples and distilled water were tested. CDV-specific bands were observed
19
with samples containing 107, 106, 105, 104 and 103 copies of CDV RNA.
20 21
In the second-round PCR, representatives of the various genetic lineages were correctly
22
characterized, yielding amplicons of the expected size (Figure 4). The reference strains
23
Accepted Manuscript
DK91B, DK91C and DK86 were correctly characterized as CDVs of the European lineage by
1
primer 200, while strains GR88, 179/04 and 48/05 were correctly recognized as Arctic-like
2
CDVs by the lineage-specific primer 202b (Figure 5). Strains KDK-1, S124C, Ac96I and P94S
3
were correctly characterized as Asia-1, and strains 007Lm, 009L and 011C were correctly
4
characterized as Asia-2 (Figure 6). The Onderstepoort-based vaccine strains (vaccines A to F
5
and our laboratory strain), as well as the Snyder Hill and Distemperoid vaccine were correctly
6
recognized by primer 201b. Similarly, the Onderstepoort and Snyder Hill strains were correctly
7
characterized by primer 201b (Figure 7). No cross-reactions were observed among heterotypic
8
primers and the various CDV strains. Fourteen field isolates were characterized as Arctic-like
9
and 22 as European.
10 11
Partial or full-length sequence analysis of the H gene of eight strains confirmed the
12
results obtained by the RT-PCR genotyping assay. Three strains were confirmed as European
13
CDVs and five were confirmed as Arctic strains (Figure 1).
14 15
4. Discussion
16
In this study, an RT-PCR genotyping system was developed, that took advantage of the
17
genetic divergence observed in the H gene among the major CDV lineages. The RT-PCR
18
genotyping system was designed on the basis of lineage-specific nucleotide polymorphisms
19
scattered over the H gene. The system was designed to allow prediction of the main genetic
20
lineages of CDV and it proved useful for the correct genotypic characterization of the genotype
21
of all vaccine strains, 17/20 laboratory reference strains representative of the various genotypes
22
and all the CDV strains collected by our laboratories over a 6-year period.
23
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1
As expected, the America-2 CDVs were not recognized by any of the genotype-specific
2
primers. Indeed, due to the genetic heterogeneity of the America-2 strains, we were unable to
3
design a primer that would recognize all the strains within this lineage. The lack of primer
4
reactivity was a pattern observed only for the America-2-like strains. Nevertheless, the
5
adoption of a negative marker is intrinsically affected by the possibility that unidentified CDV
6
strains may be mischaracterized and therefore such pattern must be interpreted with caution.
7
Accordingly, the system may be adopted to recognize unequivocally five out of the six main
8
genetic lineages of CDV, and, potentially, it could be adopted in laboratories in Europe, Asia
9
and America.
10 11
The RT-PCR sensitivity (primers 204/232b) was evaluated by using samples containing
12
known amounts of viral RNA (107 to 102 copies) and the assay was able to detect up 103 copies
13
of viral RNA. Quantification of viral loads by real-time PCR revealed that CDV RNA copies in
14
infected animals ranged between 5.91 x 105 and 4.93 x 107/µl of template in ocular swabs and
15
between 6.26 x 104 to 7.58 x 106 RNA copies/µl of template in blood samples. Amounts in
16
urine reached 2.35 x 109 RNA copies/µl of template(Elia et al., 2006). Therefore, the quantity
17
of viral RNA in biological fluids and tissues during the period of viraemia is largely beyond the
18
limits of detection of the H-gene based RT-PCR assay. Accordingly, the 204/232b RT-PCR
19
proved to be sensitive and applicable to the majority of samples collected for diagnostic
20
purpose, including ocular swabs and urine. Therefore, it would be suitable for the in vivo
21
diagnosis of CDV infection. In addition, the possibility to alleviate time-consuming
22
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procedures, such as gene cloning or direct sequencing, for precise genotype characterization
1
makes this genotyping system highly suitable for diagnostic laboratories.
2 3
Molecular characterization of measles virus (MV), another well-characterized member
4
of the genus Morbillivirus, highly related to CDV, is a key component of measles surveillance
5
and epidemiological investigations to identify the source and trace the transmission pathways
6
of the virus. In MV surveillance studies, determination of the full-length haemagglutinin gene
7
sequence is required to define correctly the genetic features of novel genotypes. According to
8
current classification scheme for MVs, the strains which differ at least by 2% nt sequence from
9
other strains in their haemagglutinin gene qualifies as a new candidate of genotype. Thus far at
10
least 23 MV genotypes have been recognized by WHO and their distribution varies
11
geographically and temporarily (Weekly Epidemiological Report, WHO, 40, 2005, 80, 347-
12
351, http:/www.who.int/wer). The initial proposal of the term ‘genotype’ to distinguish among
13
the various genetic lineages of CDV was made by Bolt et al., (1997) and it is now widely
14
accepted for defining novel CDV lineages (Mochizuki et al., 1999). However, such CDV
15
genotype definition does not correspond well to the definition of MV genotype, since the
16
genetic distance among the various CDV genotypes is greater than that observed for MV
17
genotypes. Amino acid variation between the various lineages of CDV is > 4% (32), while the
18
highest variation (> 8%) is observed between the America-1 strains, used to develop the
19
modified live CDV vaccines currently available in the market, and all the other CDV
20
genotypes (Bolt et al., 1997; Carpenter et al., 1998; Haas et al., 1997; Harder et al., 1996;
21
Iwatsuki et al., 1997; Lednicky et al., 2004; Martella et al., 2002; Martella et al., 2006;
22
Mochizuchi et al., 1999). Therefore, CDV strains appear to be more heterogeneous and to
23
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differ more from currently used vaccine than comparable MV field and vaccine strains, since
1
the H protein divergence for MV isolates and vaccine strains only reaches 3% (Rota et al.,
2
1992).
3 4
The existence of several CDV genetic lineages raises a number of questions with regard
5
to the reasons and the effects of such observed diversification. Antigenic heterogeneity has
6
been observed between America-1 and new CDVs, by using either monoclonal antibodies
7
(Blixenkrone-Møller et al., 1992; Blixenkrone-Møller et al., 1993; Harder et al., 1993; Iwatzuki
8
et al., 2000; Örvell et al., 1990) or polyclonal immune sera (Gemma et al., 1996a; Harder et al.,
9
1993; 1996; Mochizuki et al., 2002). Sera raised against wild-type CDV isolates may have
10
neutralizing titers up to tenfold higher against the homologous virus than against vaccine
11
strains (Harder et al., 1996). While it is unlikely that slight antigenic variation may
12
compromise the effectiveness of the canine vaccines in dogs, that are protected by a strong
13
active immune response elicited by repeated vaccine administrations, it is possible, however,
14
that critical amino acid substitutions in key epitopes of the H protein among circulating strains
15
may allow escape from the limited antibody repertoire of maternal origin of young
16
unvaccinated pups.
17 18
Differentiation between vaccine and field strains of CDV have relied on the use of
19
monoclonal antibodies (Iwatzuki et al., 2000; Örvell et al., 1990) and restriction or sequence
20
analysis of the full-length or partial gene H sequence (Lan et al., 2006; Mochizuki et al. 1999;
21
Ohashi et al., 1998; Uema et al. 2005). In this multicentric study we developed a genotyping
22
protocol that successfully characterised a large collection of field, laboratory and vaccine
23
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strains. Using the method described here, CDV was detected in 10/36 pups, less than 3 months
1
old, that had been recently vaccinated against CDV with ML-CDV vaccines. The pups were
2
suspected to have developed vaccine-induced disease. Samples from the pups were sent to our
3
laboratories for diagnostic analysis and all tested positive for CDV. Since the pups became ill
4
within 2 weeks after vaccine administration, the owners and some veterinarians believed that
5
the illnesses were associated with the CDV vaccines used. These resulted in disputes between
6
the veterinary practitioners and the pet owners. In each of those cases, field CDV strains were
7
found to be the cause of disease and the deaths were not related to the vaccine strains.
8 9
Interestingly, analysis of the CDV strains collected during our 2005-2006 surveillance
10
revealed the spread of Arctic-like CDVs in Italian dogs. In Italy, the Arctic strains were first
11
described in 2004 (Martella et al., 2006) and it was not clear whether this finding was
12
occasional. However, 7/14 strains in 2005 and 8/17 strains in 2006 were characterised as
13
Arctic, suggesting dissemination of this novel genotype in Italy. This pattern is similar to that
14
observed with MV endemic strains that may be subjected to rapid replacement over
15
consecutive years (Santibanez et al., 2002). Due to the paucity of the epidemiological surveys
16
and the use of different genes targeted in various studies, the global distribution of the major
17
CDV lineages is not clear. Analysis of CDV strains detected globally and from a variety of
18
host species would provide a more comprehensive understanding of the global ecology of
19
CDV.
20
21
Acknowledgments
22
The Authors are extremely grateful to Mr Donato Narcisi for his expert technical assistance.
23
Accepted Manuscript
The study was supported by a grant from the Danish Fur Breeders Research Fund and by a
1
grant of the Italian Ministry of University and Research (MIUR).
2 3
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4 5
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Appel, M.J.G., Summers, B.A., 1995. Pathogenicity of morbilliviruses for terrestrial
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Appel, M.J., Reggiardo, C., Summers, B.A., Pearce-Kelling, S., Mare, C.J., Noon, T.H.,
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Reed, R.E., Shively, J.N., Orvell. C., 1991. Canine distemper virus infection and
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encephalitis in javelinas (collared peccaries). Arch. Virol. 119, 147-152.
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Appel, M.J., Yates, R.A., Foley, G.L., Bernstein, J.J., Santinelli, S., Spelman, L.H.L.,
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Miller, D., Arp, L.H., Anderson, M., Barr, M., et al., 1994. Canine distemper epizootic in
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Accepted Manuscript
Table 1. List of the vaccine and reference CDV strains used in this study. n.r.: non reactive
1 2 3
Strain Origin Lineage/
genotype Primer reactivity Onderstepoort Vaccine A America-1 201b Onderstepoort Vaccine B America-1 201b Onderstepoort Vaccine C America-1 201b Onderstepoort Vaccine D America-1 201b Onderstepoort Vaccine E America-1 201b Snyder Hill Vaccine America-1 201b Distemperoid Vaccine America-1 201b Onderstepoort Laboratory America-1 201b Onderstepoort Laboratory America-1 201b Onderstepoort Laboratory America-1 201b Snyder Hill Laboratory America-1 201b
Dog/US89 US America-2 n.r.
Javelina/US89 US America-2 n.r.
Leopard/US91 US America-2 n.r.
Dog/GR88 Greenland Arctic 202b
48/05 Italy Arctic 202b
179/04 Italy Arctic 202b
Dog/DK91B Denmark European 200 Dog/DK91C Denmark European 200 Mink/DK86 Denmark European 200
KDK-1 Japan Asia-1 199b
S124C Japan Asia-1 199b
Ac96I Japan Asia-1 199b
P94S Japan Asia-1 199b
007Lm Japan Asia-2 203b
009L Japan Asia-2 203b
011C Japan Asia-2 203b
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Accepted Manuscript
Table 2: Sequence and position of the primers used in this study. Sense (+) and antisense (-)
1
direction is also indicated.
2 3
Primer Position (nt) Sequence 5’ to 3’
204 (+) 388-409 GAA TTC GAC TTC CGC GAT CTC C 232b (-) 1543-1519 TAG GCA ACA CCA CTA ATT TRG ACT C 199b (-) 613-554 TAG ATA CGG ATA GGG GGA AT
200 (-) 670-650 TAG CAG TTA GCA TAT TGG 201b (-) 1010-990 CAG GTA TCA CTT CCT CCA TG 202b (-) 1127-1110 TTT TTT TGT TCC TCT AGG 203b (-) 1474-1455 GAT AAC AAT TTC CAC TCG AC
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Accepted Manuscript
FIGURE LEGENDS
1 2
Figure 1: Phylogenetic relationships between CDV strains on the basis of the nucleotide
3
alignment of the H protein. Abbreviations: It, Italy; US, United States; Ch, China; Ger,
4
Germany; Dk, Denmark, Tw, Taiwan; Thai, Thailand; Tk, Turkey; Jp, Japan.
5 6
Figure 2: Scheme of the genotyping strategy used for CDV characterization.
7 8
Figure 3: RT-PCR with primer 204-232b for amplification of the 1160 bp fragment of the H
9
gene of CDV. M: 1 kb DNA ladder (Gene RulerTM, Fermentas Gmbh, Germany); lane 1:
10
Onderstepoort; lane 2: DK91B; lane 3: GR88; lane 4: KDK-1; lane 5: 007Lm; lane 6: negative
11
control.
12 13
Figure 4: Second-round PCR for prediction of CDV genotype. M: 1 kb DNA ladder (Gene
14
RulerTM, Fermentas Gmbh, Germany); lane 1: Onderstepoort (America-1); lane 2: DK91B
15
(European); lane 3: GR88 (Arctic); lane 4: KDK-1 (Asia-1); lane 5: 007Lm (Asia-2); lane 6:
16
negative control.
17 18
Figure 5: Second-round PCR for prediction of CDV genotype. Analysis of European and
19
Arctic strains. M: 100 bp DNA ladder (Gene RulerTM, Fermentas Gmbh, Germany); lane 1:
20
Onderstepoort (America-1); lane 2: DK91B (European); lane 3: DK91A (European); lane 4:
21
DK86 (European); lane 5: GR88 (Arctic); lane 6: 48/05 (Arctic); lane 7: 179/04 (Arctic).
22 23
Accepted Manuscript
Figure 6: Second-round PCR for prediction of CDV genotype. Analysis of Asia-1 and Asia-2
1
strains. 100 bp DNA ladder (Gene RulerTM, Fermentas Gmbh, Germany); lane 1:
2
Onderstepoort (America-1); lane 2: DK91B (European); lane 3: GR88 (Arctic); line 4: KDK-1
3
(Asia-1); lane 5: 007Lm (Asia-2); lane 6: S124C (Asia-1); lane 7: 009L (Asia-2); lane 8: 011C
4
(Asia-2); lane 9: Ac96I (Asia-1); line 10: P94S (Asia-1); lane 11: Snyder Hill vaccine
5
(America-1).
6 7
Figure 7: Second-round PCR for prediction of CDV genotype. Analysis of America-1
8
(vaccine) and -2 strains. 100 bp DNA ladder (Gene RulerTM, Fermentas Gmbh, Germany); lane
9
1: Onderstepoort, laboratory 1 strain (America-1); lane 2 to 6: Onderstepoort-based vaccine
10
strains, A to E, respectively; lane 7: Snyder Hill (America-1); lane 8: Distemperoid (America-
11
1); lane 9: Dog/US89 (America-2).
12
13 14
Accepted Manuscript
gi|5870826|LesserPanda/Ch gi|5870824|GiantPanda/Ch
gi|2315144|Yanaka/Jp gi|1944378|Ueno/Jp gi|6594288|KDK-1/Jp gi|3721905|TANU96/Jp
gi|1944380|Hamamatsu/Jp AB212965/S124C/Jp gi|37626037|TN/Ch gi|35187511|CDTaichung/Tw DQ887547/NTU-1/Thai/05
DQ887547/NTU/Thai/05 DQ191767/NTU-4/Thai/03 AB250943/Ac96I-p20/Jp
AB212964/P94S/Jp AB212963/Ac96I-p1/Jp
Asia-1
gi|38230168|00-2601/Raccon/Us gi|1296494|A92-27/4/Lopard/US
gi|40738381|01-2676/Raccon/Us gi|51172727|01-2689/Raccon/US gi|5733642|A75/17
gi|1296492|A92-6/Black leopard/US gi|1149601|US91/Leopard/US gi|42555995|01-2641/Raccon/US
gi|1149613|US89/Dog/US gi|1149603|javelina/US
gi|1149605|US89/Raccon/US
America-2
It-3 It-5
DQ494317/324/03/It gi|1657252|2544/Ger
gi|1657256|4513/Ger gi|1657254|404/Ger
gi|20152159|Tk 111/03A2/It 111/03B1/It 312/04/It 265/02-3
It-6
gi|1149611|DK91C/Dk gi|33320171|DK91A/Dk gi|33320183|DK91D/Dk gi|37654450|5804P/Ger gi|37654441|5804/Ger
Europe
gi|1149607|DK86/Mink/Dk DQ228166|207/00/Fox gi|693950|PDV-2Seal/Siberia gi|1149609|GR88/Dog/Greenland
gi|5733086|Liud/Dog/Ch It-4
179/04/It 48/05/It
It-7 It-1
It-2 It-8
Arctic
gi|13365646|HM-3/Jp gi|13365648|HM-6/Jp gi|5869513|98-002/Jp gi|13365644|26D/Jp gi|34328599|5VD/Jp
gi|34328597|5B/Jp AB212730/007Lm/Jp
AB252717/011C/Jp AB252718/009L/Jp
Asia-2
gi|10281214|Snyder Hill/US Snyder Hill/Vaccine gi|49182263|98-2646/Raccon/US
gi|49182261|98-2654Raccon/US gi|517242|Convac/US
Onderstepoort/VaccineA gi|14150871|Onderstepoort/US Onderstepoort/Lab3
Onderstepoort/VaccineB Onderstepoort/VaccineE Onderstepoort/VaccineD Onderstepoort/Lab2
America-1 (vaccines)
2 6 3 9 1 0 0
1 0 0
1 0 0 1 0 0
1 0 0
8 8 9 9
1 0 0 9 9 6 0 1 0 0
9 2 1 0 0
9 6 8 7 1 0 0
9 9 6 3 9 9
6 4 9 7 5 3 3 9
9 5
1 0 0
1 0 0
9 9
7 4 9 8 9 5
7 3 9 0 8 8
8 2 9 9 9 9
9 6 1 0 0
8 8 9 9 9 2
8 5 5 0
4 2 2 7
2 4 8 2
8 1 5 6 5 6
7 8
3 6
1 0 0
6 6 9 2
9 8 8 0
8 3 8 9
9 0
Accepted Manuscript
Gene H (1824 bp)
204 232b
203b 202b
201b 200
199b 1160 bp
1086 bp 739 bp 622 bp 282 bp 225 bp
(Asia 2)
(Asia 1)
(America 1, vaccines) (Europe)
(Arctic)
Figure 2
Accepted Manuscript
Figure 3
M 1 2 3 4 5 6
Accepted Manuscript
Figure 4
M 1 2 3 4 5 6
Accepted Manuscript
Figure 5
M 1 2 3 4 5 6 7
Accepted Manuscript
Figure 6
M 1 2 3 4 5 6 7 8 9 10 11
Accepted Manuscript
Figure 7
M 1 2 3 4 5 6 7 8 9
