Isolation, Tissue Distribution and Molecular

Characterization of Two Recombinant Canine

Coronavirus Strains
V. Ntafis, V. Mari, N. Decaro, M. Papanastassopoulou, N. Papaioannou, R.
Mpatziou, C. Buonavoglia, E. Xylouri

To cite this version:

V. Ntafis, V. Mari, N. Decaro, M. Papanastassopoulou, N. Papaioannou, et al.. Isolation, Tissue
Distribution and Molecular Characterization of Two Recombinant Canine Coronavirus Strains. Vet-
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Accepted Manuscript

Title: Isolation, Tissue Distribution and Molecular

Characterization of Two Recombinant Canine Coronavirus
Strains

Authors: V. Ntafis, V. Mari, N. Decaro, M.

Papanastassopoulou, N. Papaioannou, R. Mpatziou, C.
Buonavoglia, E. Xylouri

PII: S0378-1135(11)00146-5
DOI: doi:10.1016/j.vetmic.2011.03.008
Reference: VETMIC 5231

To appear in: VETMIC

Received date: 14-12-2010

Revised date: 5-3-2011
Accepted date: 10-3-2011

Please cite this article as: Ntafis, V., Mari, V., Decaro, N., Papanastassopoulou,
M., Papaioannou, N., Mpatziou, R., Buonavoglia, C., Xylouri, E., Isolation, Tissue
Distribution and Molecular Characterization of Two Recombinant Canine Coronavirus
Strains, Veterinary Microbiology (2010), doi:10.1016/j.vetmic.2011.03.008

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 1 Isolation, Tissue Distribution and Molecular Characterization of Two

2 Recombinant Canine Coronavirus Strains

4 Ntafis V.1*, Mari V.2, Decaro N.2, Papanastassopoulou M.3, Papaioannou N.4,

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5 Mpatziou R. 1, Buonavoglia C.2, Xylouri E.1

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1
7 Department of Anatomy and Physiology of Farm Animals, Faculty of Animal

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8 Science and Aquaculture, Agricultural University of Athens, Iera Odos 75, 118 55,

9 Athens, Greece.

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10 Department of Animal Health and Well-being, Faculty of Veterinary Medicine of

11 Bari, S.p. per Casamassima km 3 - 70010 Valenzano, Bari, Italy.

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12 Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine,

13 Aristotle University of Thessaloniki, 541 24, Thessaloniki, Greece.

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14 Department of Pathology, School of Veterinary Medicine, Aristotle University of
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15 Thessaloniki, 541 24, Thessaloniki, Greece.

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17
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19

20

21

22 *Corresponding author. Tel.: +30 2105294399; fax: +30 2105294388; E-mail address:

23 vasilisntafis@hotmail.com (V. Ntafis).

24

25

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26 Abstract

27 Canine coronavirus (CCoV) is an enveloped RNA virus, responsible for

28 gastrointestinal infection in dogs. To date, two different CCoV genotypes have been

29 recognized, CCoV type I and CCoV type II. Recently, CCoV type II strains of

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30 potential recombinant origin with transmissible gastroenteritis virus (TGEV) were

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31 detected and characterized as a new subtype (CCoV-IIb) of canine coronavirus, in

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32 order to be differentiated from the “classical” CCoV type II strains (CCoV-IIa). In the

33 present study, two CCoV-IIb strains were detected in the faeces and internal organs of

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34 two puppies, which died after presenting gastrointestinal symptoms. Mixed infection

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35 of both subtypes (CCoV-IIa/IIb) was detected in the faeces, while only CCoV-IIb was

36 detected in the organs. Puppies were also infected by canine parvovirus type 2 (CPV-
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37 2). Both CCoV-IIb strains were isolated on cell cultures and subjected to sequence

38 analysis and phylogeny. By means of RT-PCR and real time RT-PCR assays, tissue
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39 distribution and quantitation of viral loads took place. These cases represent the first
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40 description of tissue distribution and quantitation of CCoV-IIb strains, detected in the

41 organs. The detection of CCoV-IIa strains, which is restricted to the faeces, suggests
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42 that CCoV-IIb strains may have an advantage in disseminating throughout a dog with
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43 CPV-2 coinfection, in contrast to common enteric CCoV-IIa strains.

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45

46

47 Key words: CCoV, recombination, TGEV-like, dog, distribution, organs

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49

50

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51 Introduction

52 Canine coronavirus (CCoV; order Nidovirales, family Coronaviridae) is a

53 large, enveloped, single stranded, RNA virus responsible for enteritis in dogs (Decaro

54 and Buonavoglia, 2008). Recently, due to changes in virus classification, the virus

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55 was classified as a member of the genus Alphacoronavirus, species

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56 Alphacoronavirus-1, together with transmissible gastroenteritis virus of swine

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57 (TGEV) and feline coronavirus (FCoV) (Carstens, 2010). The genome, 27 kb in

58 length, contains two large overlapping open reading frames (ORFs), ORF1a and

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59 ORF1b which encompass the 5΄ two thirds of the genomic RNA and encode

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60 polyproteins leading to the replicase complex. The ORFs, encoding for the structural

61 spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins and the non-
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62 structural proteins (3a, 3b, 3c, 7a and 7b), are located downstream of the replicase

63 gene (Decaro and Buonavoglia, 2008).

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64 Coronaviruses are characterized by constant genetic evolution and diversity.

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65 To date, two different CCoV types have been recognized, CCoV type I (CCoV-I) and

66 CCoV type II (CCoV-II), that share significant genetic similarity with FCoV type I
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67 (FCoV-I) and FCoV type II (FCoV-II), respectively (Decaro and Buonavoglia, 2008).
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68 Moreover, in 2009, TGEV-like CCoVs of potential recombinant origin were

69 identified and characterized as a new CCoV subtype (CCoV-IIb) (Decaro et al., 2009;
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70 Erles and Brownlie, 2009; Decaro et al., 2010).

71 CCoV is the causative agent of gastroenteritis in dogs, characterized by high

72 morbidity and low mortality. Clinical signs include anorexia, lethargy, vomiting, mild

73 to severe diarrhoea (usually lasting 1-2 weeks) and occasionally death, mainly in

74 puppies. The disease is more severe in young animals (Carmichael and Binn, 1981).

75 Systemic infections are not usual; however, during the past few years, there have been

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76 reports of fatal disease, with CCoV strains detected in the enteric tract, as well as in

77 the organs (Buonavoglia et al., 2006; Decaro et al., 2009).

78 In 2010, CCoV identification, molecular characterization and sequence

79 analysis took place for the first time in Greece, regarding common enteric CCoV-II

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80 strains detected in a severe outbreak of diarrhoea in a kennel (Ntafis et al., 2010). In

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81 the current study we report the quantitation and molecular characterization of two

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82 TGEV-like CCoV strains, detected in the organs of two puppies displaying fatal

83 enteritis.

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84

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85 Materials and Methods

86 Clinical Case
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87 During summer of 2009, two dead dogs were submitted for laboratory

88 investigation. The dogs were coming from two different pet shops of Thessaloniki, a
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89 city of northern Greece. Both dogs, a 6-weeks-old Yorkshire Terrier (66/09) and a 16-
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90 weeks-old Pomeranian (68/09), presented fever, lethargy, inappetence, severe

91 haemorrhagic diarrhea and vomiting leading to death, 2 days after the onset of the
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92 symptoms. The first puppy was vaccinated with a single dose of a polyvalent vaccine
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93 against all major infectious diseases (canine distemper, infectious hepatitis, parvoviral

94 enteritis, parainfluenza and leptospirosis) 2 weeks before the symptoms, while the
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95 second one, had never been vaccinated.

96 Necropsy examination of both dogs revealed linear haemorrhages of the

97 intestinal wall, haemorrhagic enteritis and ulcerated duodenum. Sero-sanguineous

98 fluid was observed in the abdominal cavity of the Pomeranian. Lungs of both puppies

99 were congested with multiple areas of emphysema. No lesions were observed at the

100 heart. Liver of both puppies appeared enlarged, friable and yellow-brown in color

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101 with multifocal discolorated spots. Congested vessels in the dura mater of the brain

102 were also observed.

103

104 Screening for viral pathogens

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105 Samples from the faeces and the parenchymatous organs were subjected to

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106 virological investigations, using methods previously described, regarding common

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107 canine viral pathogens e.g., canine parvovirus type 2 (CPV-2) (PCR and real time

108 PCR) (Decaro et al., 2005a, Decaro et al., 2006a, Decaro et al., 2006b), canine

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109 distemper virus (CDV) (RT-PCR) (Frisk et al., 1999), canine adenovirus type 1 and

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110 type 2 (CAV-1 and CAV-2) (PCR) (Hu et al., 2001) and CCoV (RT-PCR) (Pratelli et

111 al., 1999).

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112

113 Virus isolation

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114 For virus isolation, A-72 cell line (canine fibrosarcoma) was used. The cells
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115 were grown in Dulbecco-Minimum Essential Medium (D-MEM) supplemented with

116 10% foetal bovine serum (FBS). Faecal and tissue samples were homogenized (10%
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117 w/v) in D-MEM and centrifuged at 8,000 x g for 10 min. Supernatants were treated
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118 with antibiotics (1,000 IU/ml penicillin and 100 μg/ml streptomycin) for 30 min,

119 inoculated on partially confluent A72 cell cultures and then, they were incubated at 37
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120 ºC in a 5% CO2 incubator. After an adsorption period of 30 min, D-MEM was added.

121 Cells were daily observed for cytopathic effect (cpe) of CCoV for 5 days. An

122 immunofluorescence (IF) assay was used for the detection of CCoV at the infected

123 cells. For the IF assay a 1:100 dilution of cat polyclonal serum specific for

124 Alphacoronavirus-1 and a 1:100 dilution of goat anti-cat IgG conjugated with

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125 fluorescein isothiocyanate (Sigma Aldrich, USA). Each sample was considered

126 negative after 3 passages.

127

128 CCoV characterization and quantitation

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129 RNA was extracted from faecal and organ samples of both dogs using the

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130 QIAamp Viral RNA Mini Kit and the RNeasy Mini Kit (Qiagen GmbH, Hilden,

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131 Germany), respectively. For CCoV type I and II detection and quantitation in faecal

132 and organ samples, two real time RT-PCR assays with the same sensitivity were used

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133 (Decaro et al., 2005b). Reverse transcription was performed using GeneAmp® RNA

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134 PCR (Applied Biosystems, Italy) according to the manufacturer’s instructions.

135 For the discrimination of classical (subtype IIa) and TGEV-like (subtype IIb)
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136 CCoVs, two RT-PCR assays with comparable levels of sensitivity were performed, as

137 previously described (Decaro et al., 2010). RT-PCRs with primers 20179/INS-R
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138 (CCoV-IIa) or 20179/174-268 (CCoV-IIb) were conducted using SuperScript One-

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139 Step RT-PCR for Long Templates (Invitrogen S.R.L.). In order to verify the absence

140 of TGEV strains in the samples that were positive by CCoV-IIb specific assay, an RT-
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141 PCR, able to discriminate CCoV and TGEV according to the amplicon size was used
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142 (Wesley, 1999).

143
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144 Sequencing and sequence analysis

145 The 3΄ end of the genome of the CCoV-IIb strains was amplified as previously

146 described, using viral RNA extracted from the lungs, SuperScript One-Step RT-PCR

147 for Long Templates (Invitrogen S.R.L.) and six pairs of primers, specific for

148 overlapping fragments, encompassing ORFs 2, 3a, 3b, 3c, 4, 5, 6, 7a and 7b (Decaro

149 et al., 2007). The nucleotide sequences were determined in both directions by a

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150 commercial facility (Beckman Coulter Genomics, United Kingdom). Sequence

151 assembling and analysis were carried out using the BioEdit software package (Hall,

152 1999) and the National Center for Biotechnology Information (NCBI;

153 http://www.ncbi.nlm.nih.gov) and European Molecular Biology Laboratory (EMBL;

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154 http://www.ebi.ac.uk) analysis tools. Phylogenetic analysis was conducted using

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155 MEGA4 program (Tamura et al., 2007). Phylogenetic trees, based on the amino acid

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156 sequences of S, E, M and N proteins, were elaborated using neighbor-joining method,

157 supplying a statistical support with bootstrapping over 1,000 replicates. SimPlot was

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158 used for nucleotide sequence comparison of the two strains to Alphacoronavirus-1

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159 reference strains (Lole et al., 1999). The sequences of strains 66/09 and 68/09 were

160 registered in GenBank under the accession numbers HQ450376 and HQ450377,
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161 respectively.

162
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163 Results
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164 CCoV detection, characterization and isolation

165 By means of nested PCR assay for CCoV, viral RNA was detected in faeces,
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166 lungs, spleen, kidneys, pancreas, heart, and liver of both puppies. In addition, the
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167 brain of the Pomeranian (68/09) was tested positive, while the brain of the Yorkshire

168 Terrier (66/09) was tested negative. By genotype specific real time RT-PCR assays,
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169 only CCoV-II was detected in all positive samples. CCoV-II RNA copies/μl of

170 template in the samples are shown in Table 1.

171 In the faecal samples of the two puppies, both CCoV-II subtypes were

172 detected, whilst in the organs which tested positive, only CCoV which was

173 characterized as TGEV-like (CCoV-IIb) was detected. No TGEV strains were

174 detected in the samples.

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175 The CCoV-IIb strains (66/09 and 68/09) were isolated from the lung

176 homogenates of both puppies. A-72 cells developed a cytopathic effect that consisted

177 of cell rounding and lysis of the monolayer. In addition, cells were tested positive by

178 the immunofluorescence assay. Viral titres on cell cultures were 104.25 (66/09) and 104

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179 TCID50/50 μl (68/09) at the 3rd passage.

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180

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181 Detection of other viral pathogens

182 Both puppies were tested positive for CPV-2a field strains and negative for

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183 CDV, CAV-1 and CAV-2.

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184

185 Sequencing results and phylogenetic analysis

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186 A total of 8,822 and 8,828 nucleotides were determined for strains 66/09 and

187 68/09, respectively, encompassing ORFs 2 (S protein), 3a, 3b, 3c, 4 (E protein), 5 (M
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188 protein), 6 (N protein), 7a and 7b. Alignment of the sequences with TGEV, CCoV and
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189 FCoV reference strains available in GenBank showed the highest identity to CCoV-

190 IIb reference strain 119/08 (EU924791) (98.2% and 98.9% for 66/09 and 68/09
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191 respectively). The two Greek strains shared an identity of 98%.

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192 The spike protein gene of both strains was 4,374 nucleotides long, encoding a

193 protein of 1,457 amino acids. When compared to four TGEV-like reference strains
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194 (430/07, 119/08, 174/06 and 341/05), no insertions or deletions were observed. The

195 two strains shared 97.6% aa identity to each other, while they showed the highest aa

196 identity to CCoV-IIb reference strain 119/08 (98.3%). By Simplot analysis, the two

197 strains displayed higher nucleotide conservation with the TGEV strain Purdue than

198 with the pantropic CCoV-IIa strain CB/05, at the 5΄-end of the S gene (Figure 1).

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199 Phylogenetic analysis revealed that the two Greek strains were more closely related to

200 the four CCoV-IIb reference strains detected in dogs’ organs (Figure 2a).

201 The envelope protein was found to be 82 amino acids in length, like in most

202 canine coronavirus strains and in three TGEV-like reference strains, 119/08, 174/06

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203 and 341/05, with the exception of 430/07, which is 7 amino acids shorter. The Greek

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204 strains had high amino acid identity to each other (98.7%). E protein of strains 66/09

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205 and 68/09 had the highest amino acid identity (100% and 98.7%, respectively) to the

206 CCoV-IIb strains 341/05, 119/08, and to CCoV-IIa CB/05. In the E protein,

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207 phylogenetic analysis revealed that the two strains were closely related to CCoV type

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208 II strains (Figure 2b).

209 The membrane protein (M protein) of strains 66/09 and 68/09 was found to be
M
210 260 and 262 amino acids long, respectively. Two amino acids were missing from the

211 N-terminal end of the M protein of strain 66/09 in positions 24 and 36, as it has been
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212 also observed in reference CCoV-IIb strains 174/06 and 341/05. The two strains
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213 shared high amino acid similarity (94.6%). M protein of strains 66/09 and 68/09 had

214 the highest amino acid identity to the CCoV-IIb reference strains detected in the
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215 organs (97.3% and 100%, respectively). Phylogenetic analysis of the M protein
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216 showed that the two strains were closely related to CCoV-IIa and CCoV-IIb strains

217 (Figure 2c).

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218 The N gene (nucleoprotein) was found to be 1,149 nucleotides in length,

219 coding for a polypeptide of 382 amino acids. The two proteins were 98.1% similar.

220 The amino acid sequences had the highest identity with CCoV-IIb 119/08 (98.6% and

221 99.4%, for 66/09 and 68/09, respectively). Phylogenetic analysis revealed that the two

222 Greek strains were more closely related to CCoV-II reference strains (Figure 2d).

223

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224 Discussion

225 Homologous RNA recombination consists one of the major “powers” of

226 genetic evolution and diversity, regarding coronaviruses (Woo et al., 2009). Under

227 field conditions, mixed infections are required to give rise to recombination events. So

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228 far, experimental infections of piglets (Woods and Wesley, 1992) and dogs (Larson et

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229 al., 1979) with CCoV and TGEV strains, respectively, and the fact that feline

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230 aminopeptidase N serves as a functional receptor for both CCoV and TGEV (Tresnan

231 et al., 1996), strongly suggest that the two viruses can be found growing at the same

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232 “environment” in nature, although the exact host of recombination still remains

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233 unknown.

234 A canine coronavirus strain (UCD-1) of potential recombinant origin with

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235 TGEV, was identified for the first time in the late ’90s (Wesley, 1999). Recently,

236 TGEV-like strains were reported, circulating in dogs in different countries of Europe
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237 (Decaro et al., 2010). The strains were detected in faecal samples of dogs with
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238 gastroenteritis, they were classified as the new subtype CCoV-IIb and it was

239 suggested that they were a result of recombination events, occurring at different times
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240 of these, regarding the old strain UCD-1 (Decaro et al., 2010). In the present study,
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241 sequence and phylogenetic analysis takes place for the first time in CCoV-IIb strains

242 detected in Greece.

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243 Moreover, our findings suggest that TGEV-like CCoV strains spreading to the

244 internal organs are circulating in dogs, since so far, there has been only one report in

245 Italy (Decaro et al., 2009). By means of real time RT-PCR, tissue distribution and

246 quantitation of both strains was assessed for the first time, revealing the spreading of

247 the virus to the internal organs. The CPV-2 coinfection may contribute to the

248 spreading of TGEV-like CCoV strains, since so far, they have been only detected in

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249 organs of dogs infected also with CPV-2 (Decaro et al., 2009). However, the detection

250 of CCoV-IIa strains strictly in the faeces, in both cases, suggests that CCoV-IIb may

251 have an advantage in disseminating through the dog. In the first report of TGEV-like

252 strains detected in the organs, CCoV-I was also detected strictly in the intestinal

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253 content in two cases (Decaro et al., 2009). These cases strongly suggest a difference in

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254 pathobiology of CCoV-IIb with respect to CCoV-I/IIa.

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255 By sequence and phylogenetic analysis, it was shown that both strains

256 segregate constantly with the CCoV-IIb reference strains detected in the organs of

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257 dogs. Accordingly, the strains were highly similar to TGEV in the 5΄ end of the S

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258 gene, whereas they clustered with the pantropic CCoV variant CB/05 (subtype CCoV-

259 IIa) in the E, M and N proteins. In a previous study, CCoV-IIb strains detected in the
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260 organs were found to share higher amino acid identity with CB/05 than with CCoV

261 common enteric strains, at the level of the same proteins (Decaro et al., 2009).
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262 Whether the ability of CCoV-IIb strains to spread to the organs is related to the
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263 recently recognized recombinant protein S or to the CB/05-like proteins (E, M and N)

264 needs further research. However, the S-protein “scenario” seems to be more possible,
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265 since in coronaviruses S protein mediates receptor attachment, and tissue tropism shift
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266 has been associated with mutations in the S gene (Masters, 2006).

267 In the last decade, new genotypes and subtypes of canine coronavirus have
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268 been recognized. Furthermore, a pantropic variant with the ability to cause fatal

269 systemic infection was detected (Buonavoglia et al., 2006). Previous studies revealed

270 that there are antigenic differences between CCoV-I and II (Pratelli et al., 2004). In

271 addition, antigenic differences were observed between the two subtypes, CCoV-IIa

272 and CCoV-IIb (TGEV-like CCoVs) (Decaro et al., 2009). Whether the currently

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273 circulating vaccines can protect against the TGEV-like recombinant isolates or not has

274 to be verified via vaccinations and experimental infections.

275

276 Conclusion

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277 In conclusion, this was the first report of CCoV-IIb tissue distribution. Up to

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278 now, there has been only one report of TGEV-like strains detected in internal organs

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279 of puppies in Italy. Based on sequence and phylogenetic analysis of the structural

280 proteins, the two Greek isolates were found to be related to the Italian prototype

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281 CCoV-IIb strains. In addition, in all cases a mixed infection with CPV-2 was reported.

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282 However, the detection of CCoV-IIa strains, strictly at the faeces, suggests that

283 CCoV-IIb strains may have an advantage in disseminating throughout a dog with
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284 CPV-2 coinfection, in contrast to common enteric CCoV-IIa strains.

285
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286 Acknowledgements
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287 Ntafis Vasileios is grateful to Alexander S. Onassis Public Benefit Foundation

288 for doctoral funding.

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289
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341 Lole, K.S., Bollinger, R.C., Paranjape, R.S., Gadkari, D., Kulkarni, S.S., Novak, N.G.,

342 Ingersoll, R., Sheppard, H.W., Ray, S.C., 1999. Full-length human

343 immunodeficiency virus type 1 genomes from subtype C-infected

344 seronconverters in India, with evidence of intersubtype recombination. J. Virol.

345 72, 152-160.

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346 Masters, P.S., 2006. The molecular biology of coronaviruses. Adv. Virus Res. 66:

347 193-292.

348 Ntafis, V., Mari, V., Danika, S., Fragkiadaki, E., Buonavoglia, C., 2010. An outbreak

349 of canine coronavirus in a Greek kennel. J. Vet. Diagn. Invest. 22, 320-323.

t
350 Pratelli, A., Tempesta, M., Greco, G., Martella, V., Buonavoglia, C., 1999.

ip
351 Development of a nested PCR assay for the detection of canine coronavirus. J.

cr
352 Virol. Methods 80: 11–15.

353 Pratelli, A., Elia, G., Decaro, N., Tola, S., Tinelli, A., Martella, V., Rocca, S.,

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354 Tempesta, M., Buonavoglia, C., 2004. Cloning and expression of two fragments

an
355 of the S gene of canine coronavirus type I. J. Virol. Methods 117, 61-65.

356 Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary
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357 Genetics Analysis (MEGA) Software Version 4.0. Mol. Biol. Evol. 24, 1596-

358 1599.
d

359 Tresnan, D.B., Levis, R., Holmes, K.V., 1996. Feline Aminopeptidase N Serves as a
te

360 Receptor for Feline, Canine, Porcine, and Human Coronaviruses in Serogroup I.

361 J. Virol. 70(12), 8669-8674.

p

362 Wesley, R.D., 1999. The S gene of canine coronavirus, strain UCD-1, is more closely
ce

363 related to the S gene of transmissible gastroenteritis virus than to that of feline

364 infectious peritonitis virus. Virus Res. 61, 145-152.

Ac

365 Woo, P.C.Y., Lau, S.K.P., Huang, Y., Yuen, K.Y., 2009. Coronavirus diversity,

366 phylogeny and interspecies jumping. Exp. Biol. Med. 234, 1117-27.

367 Woods, R.D., Wesley, R.D., 1992. Seroconversion of pigs in contact with dogs

368 exposed to canine coronavirus. Can. J. Vet. Res. 56, 78-80.

369

370

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371 Figure captions

372 Figure 1. S gene sequences analysis with Simplot. The S gene of CCoV-IIb strain

373 68/09, TGEV strain Purdue and CCoV-IIa pantropic strain CB/05 were plotted against

374 the S gene of CCoV-IIb strain 66/09.

t
375 Figure 2. Neighbor-joining trees of the Greek strains, based on the S (a), E (b), M (c)

ip
376 and N (d) protein. The trees are rooted on the group 2 canine respiratory coronavirus

cr
377 (CRCoV). The numbers represent the percentage of replicate trees based on 1,000

378 bootstrap replicates.

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Table 1

Tables

Table 1. CCoV-II RNA copies/μl of template in the samples of the two puppies, tested

by genotype-specific real time RT-PCR.

66/09 68/09
Sample

t
(Yorkshire Terrier) (Pomeranian)

ip
Faeces 3.59 x 103 7.22 x 105

cr
Liver 4.64 x 104 3.21 x 105

us
Spleen 5.20 x 105 1.55 x 107

Pancreas 2.75 x 102 2.03 x 104

Kidney 1.23 x 105

an 3.37 x 106

Lung 5.99 x 106 4.10 x 106

M
Heart 1.14 x 105 7.08 x 106

Brain n.d. 2.47 x 103

ed

n.d., not detected

pt
ce
Ac

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Figure 1

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Figure 2a

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Figure 2b

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Figure 2c

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Figure 2d

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