SHORT COMMUNICATION Occurrence of different Canine distemper virus lineages in Italian dogs Andrea Balboni*, Giorgia De Lorenzo Dandola, Alessandra Scagliarini, Santino Prosperi & Mara Battilani Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell’Emilia (BO), Italy * Corresponding author at: Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell’Emilia (BO), Italy. Tel.: +39 051 2097084, e-mail: [email protected].
Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2 Accepted: 04.09.2014 | Available on line: 30.09.2014
Keywords Arctic-like, Canine distemper virus, Dog, Europe 1, Gene H, Italy.
Summary This study describes the sequence analysis of the H gene of 7 Canine distemper virus (CDV) strains identified in dogs in Italy between years 2002-2012. The phylogenetic analysis showed that the CDV strains belonged to 2 clusters: 6 viruses were identified as Arctic‑like lineage and 1 as Europe 1 lineage. These data show a considerable prevalence of Arctic‑like‑CDVs in the analysed dogs. The dogs and the 3 viruses more recently identified showed 4 distinctive amino acid mutations compared to all other Arctic CDVs.
Presenza di diversi lineage di Canine distemper virus in cani in Italia Parole chiave Arctic-like CDV, Cane, Canine distemper virus (CDV), Europe 1 CDV, Gene H, Italia.
Riassunto L’articolo riporta l’analisi della sequenza del gene H di 7 ceppi di Canine distemper virus (CDV) identificati in cani, in Italia, tra il 2002 e il 2012. L’analisi filogenetica ha mostrato l’appartenenza dei 7 ceppi a 2 gruppi genetici: 6 ceppi al lineage Arctic-like e un ceppo al lineage Europe 1. I dati raccolti hanno mostrato una considerevole prevalenza nei cani testati di CDV appartenenti al lineage Arctic-like, evidenziando negli ultimi 3 ceppi virali identificati la presenza di 4 mutazioni aminoacidiche distintive rispetto agli altri ceppi di Arctic-like CDV.
Currently, 9 genetic lineages of Canine distemper virus (CDV) are recognized throughout the world: America 1, America 2, Asia 1, Asia 2, Europe Wildlife, Arctic-like, South Africa, Europe 1/South America 1 and South America 2. In addition, new CDV lineages were recently proposed to include viruses circulating in Asia (Asia 3) and viral strains related to Rockborn vaccine strain (Calderon et al. 2007, Martella et al. 2011, Panzera et al. 2012, Zhao et al. 2010). The attachment/haemagglutinin (H) is the most variable CDV protein and has been used throughout the years to distinguish the different genetic lineages and to classify the CDV circulating strains. Furthermore, the 3' end of H gene codifies for important amino acid domains involved in interaction between virus and cellular receptor and it has been hypothesized that residues 530 and 549 might affect the viral tropism (McCarthy et al. 2007, Nikolin et al. 2012). The CDV-related disease in canine population
has generally been controlled by live attenuated vaccines, but outbreaks of CDV are still reported worldwide also in vaccinated animals (Martella et al. 2007). Moreover, sequencing of several CDV strains belonging to different genetic lineages has highlighted considerable genetic and antigenic diversities (especially in the H gene/protein). However the effects that these mutations may have on the virulence remain unclear, as well as the susceptibility of different carnivore hosts and the ability of the currently available vaccines to protect from infection (McCarthy et al. 2007, Nikolin et al. 2012, Sekulin et al. 2011). Three CDV genetic lineages are currently circulating in Italy: the Europe 1 lineage (historically spread in the dogs as well as in wild animals), also named Europe 1/South America 1 because it also circulates in South America, the Europe Wildlife (prevalently diffused in wildlife and sporadically reported in
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dogs) and the Arctic-like lineage (native to the Arctic ecosystem and recently detected in Italian dogs) (Martella et al. 2006, Martella et al. 2007, Monne et al. 2011, Panzera et al. 2012). Furthermore, a distinct viral subgroup insight the Europe 1 lineage (named Wildlife Europe 2006-2009) has been shown to be responsible for the recent epidemic in Alpine wildlife, both in Italy and in Central Europe (Monne et al. 2011, Sekulin et al. 2011). Until the year 2000, only the Europe 1 lineage had been circulating in the Italian dog population. Instead, after the year 2000, several cases of Arctic-like CDVs infection were reported in dogs. Between 2002 and 2006, Martella and collaborators revealed cases of Arctic-like CDVs infection in Central and Southern Italy (Martella et al. 2006, Martella et al. 2007) and, between 2000 and 2007, cases of Arctic-like CDVs infection were also reported in the North-Eastern Italy (Monne et al. 2011). In contrast, a survey conducted on 53 dogs with symptoms attributable to CDV infection from Central Italy only identified CDV strains belonging to the classical Europe 1 lineage (Di Francesco et al. 2012). It is therefore yet to be understood if the genotype Arctic-like is permanently established in the Italian canine population or if it represents occasional findings in certain geographic areas. In order to get further information on the CDV strains circulating in Italy, 7 CDV strains from Emilia Romagna and Lazio were analysed. They were capable of causing clinical signs in infected dogs as referred by local veterinary surgeons (Table I). With this aim, viral RNA was extracted from the footpad or brain using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and molecular diagnosis of CDV was done by TaqMan based real-time polymerase chain reaction (RT-PCR) (Scagliarini et al. 2007). The CDV H gene was amplified using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) and the set of primers C previously described by Demeter and colleagues (Demeter et al. 2007). The extremity 3’ of H gene was directly sequenced obtaining a fragment of 582 bp in length, corresponding to the
last 193 amino acid residues of the H protein. The obtained nucleotide sequences were aligned and compared with 119 reference sequences available from the GenBank database1 using the CLUSTALW software implemented in BioEdit sequence alignment editor version 7.0.9. The phylogenetic relationships were evaluated using MEGA version 5.05, with the best-fit model of nucleotide substitution determined using the function Find Best DNA/Protein Model implemented in the program. Pairwise genetic distances were calculated by Tamura-Nei model with γ distribution that resulted optimal for all the sequence data and phylogenetic trees were constructed using the neighbor-joining method. Bootstrap values were determined by 1000 replicates to assess the confidence level of each branch pattern. The nucleotide sequences obtained have been lodged within the GenBank sequence database under accession numbers: KF184985KF184991. The nucleotide sequences demonstrate that the analysed CDV strains belonged to 2 clusters. The first cluster included 6 Arctic-like lineage viruses (444.2002, 456.2003, 64.2004, 99.2011, 319.2012 and 352.2012) that showed an identity of 97.7‑99.8% among themselves and of 97.2-100% with other Arctic-like strains. In particular, 444.2002 showed a complete identity with 3 North Italian CDVs identified in dogs in 2000-2001 (HM443711, HM443719 and HM443720). The last identified CDV strain (741.2006) belonged to Europe 1 lineage, showing an identity of 93.2-94.8% with viruses of the first cluster and of 95.7-99.8% with Europe 1 strains. Notably, the nucleotide identity calculated by comparing the viruses of the first cluster with Artic-like reference strains decreases progressively from oldest samples to samples collected more recently. Furthermore, the identities between 741.2006 and 99.2011, 319.2012 and 352.2012 were found to be lower than those calculated with the Arctic-like reference strains.
1
www.ncbi.nlm.nih.gov/genbank/.
Table I. Details of 7 CDV strains identified in dogs in Italy between years 2002-2012. Virus 444 456 64 741 99 319 352
Sampling date 2002 2003 2004 2006 2011 2012 2012
Breed Labrador retriever German shepard Cane Corso Mixed-breed Mixed-breed Mixed-breed Mixed-breed
Sex M F F Un F M F
Age 2m 2m 1y 3m Un 5m 2m 2m
Origin Emilia-Romagna Emilia-Romagna Emilia-Romagna Emilia-Romagna Lazio Lazio Lazio
Vaccination status Yes No Yes Un Un Un No
Symptoms R+N R+N GI + N + C Un GI + N GI + N N
M = male; F = female; m = months; y = years; R = respiratory symptoms; N = neurological symptoms; GI = gastrointestinal symptoms; C = cutaneous lesions; Un = unknown.
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DE_badger_2008_FJ416338 DE_fox_2008_FJ416339 DE_fox_2008_FJ416336 DE_fox_2008_FJ416337 IT_badger_2008_HM443704 CH_lynx_2010_JF810109 IT_fox_2007_HM443705 CH_fox_2010_JF810107 CH_dog_2010_JF810111 IT_fox_2009_HM120874 CH_marten_2002_JF810110 86 CH_fox_2010_JF810108 CH_fox_2010_JF810106 IT_fox_2006_HM443726 IT_badger_2006_HM443725 99 IT_fox_2009_HM443709 IT_marten_2009_HM443708 IT_fox_2008_HM443707 HU_dog_2004_DQ889177 DE_fox_2007_JN153024 91 DE_fox_2007_JN153025 100 ES_lynx_2005_GU001863 ES_marten_2005_GU001864 PT_wolf_1998_HM563057 100 PT_wolf_2008_HM563058 PT_dog_2007_HM563059 AU_dog_2002_GQ214376 AU_dog_2002_GQ214378 97 AU_dog_2002_GQ214380 AU_dog_2002_GQ214384 IT_dog_2003_DQ494319 IT_dog_741.2006_KF184988 97 IT_dog_2002_DQ494318 IT_dog_2007_HM443723 DK_dog_1994_Z47761 89 IT_dog_2000_HM443718 TR_dog_2002_AY093674 IT_dog_2003_DQ494317 DE_dog_1996_Z77671 DE_dog_1996_Z77672 DE_dog_1996_Z77673 AR_dog_2003_FJ392652 UY_dog_2008_JN215474 99 UY_dog_2007_JN215473 99 UY_dog_2008_JN215475 UY_dog_2009_JN215476 UY_dog_2009_JN215477 US_raccoon_2001_AY498692 US_dog_1995_Z47762 US_raccoon_2001_AY526496 US_bleopard_1992_Z47763 CN_gpanda_1999_AF178038 JP_dog_2005_AB212965 JP_dog_1996_D85754 98 JP_badger_2007_AB329581 CN_dog_2008_FJ409464 CN_fox_2006_EU325722 CN_fox_2006_HQ540293 AR_dog_2003_AM422846 98 AR_dog_2003_AM422847 AR_dog_2005_FJ392651 US_dog_2004_AY964110 Rockborn-Candur_GU266280 US_dog_2004_AY964114 95 Rockborn-46th_GU810819 CN_lesser-panda_1999_AF178039 IT_fox_2000_DQ228166 DK_mink_1992_Z47759 HU_dog_2006_DQ889188 100 HU_dog_2006_DQ889189 HU_dog_2006_DQ889187 100 AU_badger_2006_GQ214374 AU_marten_2007_GQ214369 DE_raccoon_2007_JN153022 DE_raccoon_2007_JN153019 100 DE_raccoon_2007_JN153020 DE_raccoon_2007_JN153021 DE_raccoon_2007_JN153023 ZA_dog_2007_FJ461714 ZA_dog_2007_FJ461698 99 ZA_dog_2007_FJ461720 ZA_dog_2007_FJ461724 GL_dog_1992_Z47760 IT_dog_2000_HM443710 IT_dog_2000_HM443713 97 87 IT_dog_444.2002_KF184985 IT_dog_2000_HM443720 93 IT_dog_2000_HM443719 IT_dog_2001_HM443711 IT_dog_2000_HM443716 IT_dog_2000_HM443717 IT_dog_2000_HM443714 US_dog_2004_AY964112 US_dog_2004_AY964108 IT_dog_2005_HM443712 IT_dog_2005_DQ226088 IT_dog_99.2011_KF184989 98 IT_dog_319.2012_KF184990 88 IT_dog_352.2012_KF184991 IT_dog_2008_HM443706 IT_dog_2002_HM443721 IT_dog_2004_HM443715 IT_dog_2004_DQ226087 84 IT_dog_64.2004_KF184987 IT_dog_456.2003_KF184986 IT_dog_2002_HM443724 AU_dog_2003_GQ214373 IT_dog_2002_HM443722 HU_dog_2005_DQ889181 HU_dog_2005_DQ889178 HU_dog_2005_DQ889179 HU_dog_2005_DQ889180 HU_dog_2005_DQ889182 HU_dog_2005_DQ889183 HU_dog_2006_DQ889184 HU_dog_2006_DQ889185 HU_dog_2006_DQ889186 CN_fox_2005_EU743935 JP_dog_1998_AB025270 97 JP_dog_2006_AB252718 91 Onderstepoort_AF305419 Convac_Z35493 Lederle_EF418782 99 SnyderHill_AF259552 99 US_raccoon_1998_AY548109 PDV-1_AF479277
A Europe 1
South America Proposed Lineage America II Asia I
South America Proposed Lineage Rockborn-like Proposed Lineage
Europe Wildlife
South Africa
Arctic-like
Asia II America I
0.05
Figure 1. Rooted phylogenetic tree constructed on nucleotide sequences of the 3’ fragment of H gene of the CDV genome (582 base pairs). The phylogenetic tree was constructed using the neighbor-joining method with the nucleotide sequences generated in this study and with sequences of 118 CDV reference strains obtained from the GenBank database and a Phocine distemper virus as outgroup. Bootstrap values greater than 80% are indicated on the respective branches. The CDV strains included in the phylogenetic analysis are named with: acronym of nation, host species and year of identification (with lab numbers for sequences generated in this study), plus the GenBank accession number. When the year of identification was not available, the year of deposition of the nucleotide sequence in the GenBank database was indicated. Highlighted in black: sequences generated in this study. Highlighted in gray: Wildlife Europe 2006-2009 subgroup.
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These findings are further supported by the phylogenetic tree, which depicts 6 viruses clustering in the Arctic-like lineage and 741.2006 in the Europe 1/South America 1 lineage (Figure 1). Furthermore, 99.2011, 319.2012 and 352.2012, formed a monophyletic clade inside the Arctic-like lineages, strictly related with a North Italian CDV identified in 2008 (HM443706). The H protein of the 6 Arctic-like CDVs exhibited residues asparagine (N) at position 530 and tyrosine (Y) at position 549, in accordance with all Arctic-like strains sequenced until now; whereas, in accordance with all Europe 1 strains identified in domestic dogs, 741.2006 exhibited residues glycine (G) at position 530 and tyrosine (Y) at position 549 (Martella et al. 2007, Monne et al. 2011). Some differences were detected in other amino acid positions, as shown in Figure 2. In particular, 99.2011, 319.2012 and 352.2012 exhibit methionine (M) at position 445 and serine (S) at position 559 which were never reported previously in other CDVs. Furthermore, these latter Arctic-like viruses exhibit threonine (T) at position 417 and asparagine (N) at position 435 previously reported in another North Italian CDV strain HM443706.
These data show a considerable spread of Arctic‑like lineage in the sampled dogs, in agreement with previous reports on CDV strains collected in Italy which had revealed the presence of Arctic-like CDVs in dogs since 2000 and confirm the dissemination of this novel genotype in Italy (Martella et al. 2006, Martella et al. 2007, Monne et al. 2011). The Arctic‑like lineage is native to the Arctic ecosystem and was usually related to infection of wild animals; it was only occasionally associated with outbreaks in domestic dogs from country geographically distant from Italy, such as Hungary, United States of America, China and Greenland (Blixenkrone‑Möller et al. 1992, Demeter et al. 2007, Pardo et al. 2005). Subsequently to the arrival of Arctic-like CDVs in Italy, probably due to a significant movement of viruses from the East‑Central Europe to Italy in consequence of intense trade of dogs (Martella et al. 2006), the increasing detection of these viruses might suggest that strains belonging to Arctic‑like lineage are becoming progressively endemic in Italy. Furthermore, the 3 viruses more recently identified (99.2011, 319.2012 and 352.2012) showed 4 distinctive amino acid mutations compared to all other Arctic CDVs. Although the study shows a high prevalence of Arctic-like CDV in sampled dogs, the number of viral
Figure 2. Amino acids differences in the sequence tract of H gene between the 7 viral strains identified in dogs in Italy between years 2002-2012. The ruler at the top shows the amino acid positions corresponding to the entire H protein.
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strains analysed should be increased, extending the monitoring activities also to other geographical areas, to properly assess the incidence of Arctic-like CDV strains in Italian dogs. Further studies would
Canine distemper virus in Italian dogs
be necessary to understand what benefits the new amino acid mutations can confer to the Arcticlike CDVs and in which way these may affect their spreading in Italy and in Europe.
References Blixenkrone-Möller M., Svansson V., Appel M., Krogsrud J., Have P. & Orvell C. 1992. Antigenic relationships between field isolates of morbilliviruses from different carnivores. Arch Virol, 123(3-4), 279-294. Calderon M.G., Remorini P., Periolo O., Iglesias M., Mattion N. & La Torre J. 2007. Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina. Vet Microbiol, 125(3-4), 341-349. Demeter Z., Lakatos B., Palade E.A., Kozma T., Forgách P. & Rusvai M. 2007. Genetic diversity of Hungarian canine distemper virus strains. Vet Microbiol, 122(3-4), 258-269. Di Francesco C.E., Di Francesco D., Di Martino B., Speranza R., Santori D., Boari A. & Marsilio F. 2012. Detection by hemi-nested reverse transcription polymerase chain reaction and genetic characterization of wild type strains of Canine distemper virus in suspected infected dogs. J Vet Diagn Invest, 24(1), 107-115. Martella V., Blixenkrone-Møller M., Elia G., Lucente M.S., Cirone F., Decaro N., Nielsen L., Bányai K., Carmichael L.E. & Buonavoglia C. 2011. Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain. Vaccine, 29(6), 1222-1227. Martella V., Cirone F., Elia G., Lorusso E., Decaro N., Campolo M., Desario C., Lucente M.S., Bellacicco A.L., Blixenkrone-Møller M., Carmichael L.E. & Buonavoglia C. 2006. Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy. Vet Microbiol, 116(4), 301-309. Martella V., Elia G., Lucente M.S., Decaro N., Lorusso E., Banyai K., Blixenkrone-Møller M., Lan N.T., Yamaguchi R., Cirone F., Carmichael L.E. & Buonavoglia C. 2007. Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks. Vet Microbiol, 122(1-2), 32-42. McCarthy A.J., Shaw M.A. & Goodman S.J. 2007. Pathogen
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evolution and disease emergence in carnivores. Proc Biol Sci, 274(1629), 3165-3174. Monne I., Fusaro A., Valastro V., Citterio C., Dalla Pozza M., Obber F., Trevisiol K., Cova M., De Benedictis P., Bregoli M., Capua I. & Cattoli G. 2011. A distinct CDV genotype causing a major epidemic in Alpine wildlife. Vet Microbiol, 150(1-2), 63-69. Nikolin V.M., Wibbelt G., Michler F.U., Wolf P. & East M.L. 2012. Susceptibility of carnivore hosts to strains of canine distemper virus from distinct genetic lineages. Vet Microbiol, 156(1-2), 45-53. Panzera Y., Calderón M.G., Sarute N., Guasco S., Cardeillac A., Bonilla B., Hernández M., Francia L., Bedó G., La Torre J. & Pérez R. 2012. Evidence of two co-circulating genetic lineages of canine distemper virus in South America. Virus Res, 163(1), 401-494. Pardo I.D., Johnson G.C. & Kleiboeker S.B. 2005. Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America. J Clin Microbiol, 43(10), 5009-5017. Scagliarini A., Dal Pozzo F., Gallina L., Vaccari F. & Morganti L. 2007. TaqMan based real time PCR for the quantification of canine distemper virus. Vet Res Commun, 31(Suppl 1), 261-263. Sekulin K., Hafner-Marx A., Kolodziejek J., Janik D., Schmidt P. & Nowotny N. 2011. Emergence of canine distemper in Bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein. Vet J, 187(3), 399-401. Zhao J.J., Yan X.J., Chai X.L., Martella V., Luo G.L., Zhang H.L., Gao H., Liu Y.X., Bai X., Zhang L., Chen T., Xu L., Zhao C.F., Wang F.X., Shao X.Q., Wu W. & Cheng S.P. 2010. Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes, raccoon dogs and minks in China. Vet Microbiol, 140(1-2), 34-42.
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Keywords Arctic-like, Canine distemper virus, Dog, Europe 1, Gene H, Italy.
Summary This study describes the sequence analysis of the H gene of 7 Canine distemper virus (CDV) strains identified in dogs in Italy between years 2002-2012. The phylogenetic analysis showed that the CDV strains belonged to 2 clusters: 6 viruses were identified as Arctic‑like lineage and 1 as Europe 1 lineage. These data show a considerable prevalence of Arctic‑like‑CDVs in the analysed dogs. The dogs and the 3 viruses more recently identified showed 4 distinctive amino acid mutations compared to all other Arctic CDVs.
Presenza di diversi lineage di Canine distemper virus in cani in Italia Parole chiave Arctic-like CDV, Cane, Canine distemper virus (CDV), Europe 1 CDV, Gene H, Italia.
Riassunto L’articolo riporta l’analisi della sequenza del gene H di 7 ceppi di Canine distemper virus (CDV) identificati in cani, in Italia, tra il 2002 e il 2012. L’analisi filogenetica ha mostrato l’appartenenza dei 7 ceppi a 2 gruppi genetici: 6 ceppi al lineage Arctic-like e un ceppo al lineage Europe 1. I dati raccolti hanno mostrato una considerevole prevalenza nei cani testati di CDV appartenenti al lineage Arctic-like, evidenziando negli ultimi 3 ceppi virali identificati la presenza di 4 mutazioni aminoacidiche distintive rispetto agli altri ceppi di Arctic-like CDV.
Currently, 9 genetic lineages of Canine distemper virus (CDV) are recognized throughout the world: America 1, America 2, Asia 1, Asia 2, Europe Wildlife, Arctic-like, South Africa, Europe 1/South America 1 and South America 2. In addition, new CDV lineages were recently proposed to include viruses circulating in Asia (Asia 3) and viral strains related to Rockborn vaccine strain (Calderon et al. 2007, Martella et al. 2011, Panzera et al. 2012, Zhao et al. 2010). The attachment/haemagglutinin (H) is the most variable CDV protein and has been used throughout the years to distinguish the different genetic lineages and to classify the CDV circulating strains. Furthermore, the 3' end of H gene codifies for important amino acid domains involved in interaction between virus and cellular receptor and it has been hypothesized that residues 530 and 549 might affect the viral tropism (McCarthy et al. 2007, Nikolin et al. 2012). The CDV-related disease in canine population
has generally been controlled by live attenuated vaccines, but outbreaks of CDV are still reported worldwide also in vaccinated animals (Martella et al. 2007). Moreover, sequencing of several CDV strains belonging to different genetic lineages has highlighted considerable genetic and antigenic diversities (especially in the H gene/protein). However the effects that these mutations may have on the virulence remain unclear, as well as the susceptibility of different carnivore hosts and the ability of the currently available vaccines to protect from infection (McCarthy et al. 2007, Nikolin et al. 2012, Sekulin et al. 2011). Three CDV genetic lineages are currently circulating in Italy: the Europe 1 lineage (historically spread in the dogs as well as in wild animals), also named Europe 1/South America 1 because it also circulates in South America, the Europe Wildlife (prevalently diffused in wildlife and sporadically reported in
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dogs) and the Arctic-like lineage (native to the Arctic ecosystem and recently detected in Italian dogs) (Martella et al. 2006, Martella et al. 2007, Monne et al. 2011, Panzera et al. 2012). Furthermore, a distinct viral subgroup insight the Europe 1 lineage (named Wildlife Europe 2006-2009) has been shown to be responsible for the recent epidemic in Alpine wildlife, both in Italy and in Central Europe (Monne et al. 2011, Sekulin et al. 2011). Until the year 2000, only the Europe 1 lineage had been circulating in the Italian dog population. Instead, after the year 2000, several cases of Arctic-like CDVs infection were reported in dogs. Between 2002 and 2006, Martella and collaborators revealed cases of Arctic-like CDVs infection in Central and Southern Italy (Martella et al. 2006, Martella et al. 2007) and, between 2000 and 2007, cases of Arctic-like CDVs infection were also reported in the North-Eastern Italy (Monne et al. 2011). In contrast, a survey conducted on 53 dogs with symptoms attributable to CDV infection from Central Italy only identified CDV strains belonging to the classical Europe 1 lineage (Di Francesco et al. 2012). It is therefore yet to be understood if the genotype Arctic-like is permanently established in the Italian canine population or if it represents occasional findings in certain geographic areas. In order to get further information on the CDV strains circulating in Italy, 7 CDV strains from Emilia Romagna and Lazio were analysed. They were capable of causing clinical signs in infected dogs as referred by local veterinary surgeons (Table I). With this aim, viral RNA was extracted from the footpad or brain using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and molecular diagnosis of CDV was done by TaqMan based real-time polymerase chain reaction (RT-PCR) (Scagliarini et al. 2007). The CDV H gene was amplified using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA) and the set of primers C previously described by Demeter and colleagues (Demeter et al. 2007). The extremity 3’ of H gene was directly sequenced obtaining a fragment of 582 bp in length, corresponding to the
last 193 amino acid residues of the H protein. The obtained nucleotide sequences were aligned and compared with 119 reference sequences available from the GenBank database1 using the CLUSTALW software implemented in BioEdit sequence alignment editor version 7.0.9. The phylogenetic relationships were evaluated using MEGA version 5.05, with the best-fit model of nucleotide substitution determined using the function Find Best DNA/Protein Model implemented in the program. Pairwise genetic distances were calculated by Tamura-Nei model with γ distribution that resulted optimal for all the sequence data and phylogenetic trees were constructed using the neighbor-joining method. Bootstrap values were determined by 1000 replicates to assess the confidence level of each branch pattern. The nucleotide sequences obtained have been lodged within the GenBank sequence database under accession numbers: KF184985KF184991. The nucleotide sequences demonstrate that the analysed CDV strains belonged to 2 clusters. The first cluster included 6 Arctic-like lineage viruses (444.2002, 456.2003, 64.2004, 99.2011, 319.2012 and 352.2012) that showed an identity of 97.7‑99.8% among themselves and of 97.2-100% with other Arctic-like strains. In particular, 444.2002 showed a complete identity with 3 North Italian CDVs identified in dogs in 2000-2001 (HM443711, HM443719 and HM443720). The last identified CDV strain (741.2006) belonged to Europe 1 lineage, showing an identity of 93.2-94.8% with viruses of the first cluster and of 95.7-99.8% with Europe 1 strains. Notably, the nucleotide identity calculated by comparing the viruses of the first cluster with Artic-like reference strains decreases progressively from oldest samples to samples collected more recently. Furthermore, the identities between 741.2006 and 99.2011, 319.2012 and 352.2012 were found to be lower than those calculated with the Arctic-like reference strains.
1
www.ncbi.nlm.nih.gov/genbank/.
Table I. Details of 7 CDV strains identified in dogs in Italy between years 2002-2012. Virus 444 456 64 741 99 319 352
Sampling date 2002 2003 2004 2006 2011 2012 2012
Breed Labrador retriever German shepard Cane Corso Mixed-breed Mixed-breed Mixed-breed Mixed-breed
Sex M F F Un F M F
Age 2m 2m 1y 3m Un 5m 2m 2m
Origin Emilia-Romagna Emilia-Romagna Emilia-Romagna Emilia-Romagna Lazio Lazio Lazio
Vaccination status Yes No Yes Un Un Un No
Symptoms R+N R+N GI + N + C Un GI + N GI + N N
M = male; F = female; m = months; y = years; R = respiratory symptoms; N = neurological symptoms; GI = gastrointestinal symptoms; C = cutaneous lesions; Un = unknown.
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DE_badger_2008_FJ416338 DE_fox_2008_FJ416339 DE_fox_2008_FJ416336 DE_fox_2008_FJ416337 IT_badger_2008_HM443704 CH_lynx_2010_JF810109 IT_fox_2007_HM443705 CH_fox_2010_JF810107 CH_dog_2010_JF810111 IT_fox_2009_HM120874 CH_marten_2002_JF810110 86 CH_fox_2010_JF810108 CH_fox_2010_JF810106 IT_fox_2006_HM443726 IT_badger_2006_HM443725 99 IT_fox_2009_HM443709 IT_marten_2009_HM443708 IT_fox_2008_HM443707 HU_dog_2004_DQ889177 DE_fox_2007_JN153024 91 DE_fox_2007_JN153025 100 ES_lynx_2005_GU001863 ES_marten_2005_GU001864 PT_wolf_1998_HM563057 100 PT_wolf_2008_HM563058 PT_dog_2007_HM563059 AU_dog_2002_GQ214376 AU_dog_2002_GQ214378 97 AU_dog_2002_GQ214380 AU_dog_2002_GQ214384 IT_dog_2003_DQ494319 IT_dog_741.2006_KF184988 97 IT_dog_2002_DQ494318 IT_dog_2007_HM443723 DK_dog_1994_Z47761 89 IT_dog_2000_HM443718 TR_dog_2002_AY093674 IT_dog_2003_DQ494317 DE_dog_1996_Z77671 DE_dog_1996_Z77672 DE_dog_1996_Z77673 AR_dog_2003_FJ392652 UY_dog_2008_JN215474 99 UY_dog_2007_JN215473 99 UY_dog_2008_JN215475 UY_dog_2009_JN215476 UY_dog_2009_JN215477 US_raccoon_2001_AY498692 US_dog_1995_Z47762 US_raccoon_2001_AY526496 US_bleopard_1992_Z47763 CN_gpanda_1999_AF178038 JP_dog_2005_AB212965 JP_dog_1996_D85754 98 JP_badger_2007_AB329581 CN_dog_2008_FJ409464 CN_fox_2006_EU325722 CN_fox_2006_HQ540293 AR_dog_2003_AM422846 98 AR_dog_2003_AM422847 AR_dog_2005_FJ392651 US_dog_2004_AY964110 Rockborn-Candur_GU266280 US_dog_2004_AY964114 95 Rockborn-46th_GU810819 CN_lesser-panda_1999_AF178039 IT_fox_2000_DQ228166 DK_mink_1992_Z47759 HU_dog_2006_DQ889188 100 HU_dog_2006_DQ889189 HU_dog_2006_DQ889187 100 AU_badger_2006_GQ214374 AU_marten_2007_GQ214369 DE_raccoon_2007_JN153022 DE_raccoon_2007_JN153019 100 DE_raccoon_2007_JN153020 DE_raccoon_2007_JN153021 DE_raccoon_2007_JN153023 ZA_dog_2007_FJ461714 ZA_dog_2007_FJ461698 99 ZA_dog_2007_FJ461720 ZA_dog_2007_FJ461724 GL_dog_1992_Z47760 IT_dog_2000_HM443710 IT_dog_2000_HM443713 97 87 IT_dog_444.2002_KF184985 IT_dog_2000_HM443720 93 IT_dog_2000_HM443719 IT_dog_2001_HM443711 IT_dog_2000_HM443716 IT_dog_2000_HM443717 IT_dog_2000_HM443714 US_dog_2004_AY964112 US_dog_2004_AY964108 IT_dog_2005_HM443712 IT_dog_2005_DQ226088 IT_dog_99.2011_KF184989 98 IT_dog_319.2012_KF184990 88 IT_dog_352.2012_KF184991 IT_dog_2008_HM443706 IT_dog_2002_HM443721 IT_dog_2004_HM443715 IT_dog_2004_DQ226087 84 IT_dog_64.2004_KF184987 IT_dog_456.2003_KF184986 IT_dog_2002_HM443724 AU_dog_2003_GQ214373 IT_dog_2002_HM443722 HU_dog_2005_DQ889181 HU_dog_2005_DQ889178 HU_dog_2005_DQ889179 HU_dog_2005_DQ889180 HU_dog_2005_DQ889182 HU_dog_2005_DQ889183 HU_dog_2006_DQ889184 HU_dog_2006_DQ889185 HU_dog_2006_DQ889186 CN_fox_2005_EU743935 JP_dog_1998_AB025270 97 JP_dog_2006_AB252718 91 Onderstepoort_AF305419 Convac_Z35493 Lederle_EF418782 99 SnyderHill_AF259552 99 US_raccoon_1998_AY548109 PDV-1_AF479277
A Europe 1
South America Proposed Lineage America II Asia I
South America Proposed Lineage Rockborn-like Proposed Lineage
Europe Wildlife
South Africa
Arctic-like
Asia II America I
0.05
Figure 1. Rooted phylogenetic tree constructed on nucleotide sequences of the 3’ fragment of H gene of the CDV genome (582 base pairs). The phylogenetic tree was constructed using the neighbor-joining method with the nucleotide sequences generated in this study and with sequences of 118 CDV reference strains obtained from the GenBank database and a Phocine distemper virus as outgroup. Bootstrap values greater than 80% are indicated on the respective branches. The CDV strains included in the phylogenetic analysis are named with: acronym of nation, host species and year of identification (with lab numbers for sequences generated in this study), plus the GenBank accession number. When the year of identification was not available, the year of deposition of the nucleotide sequence in the GenBank database was indicated. Highlighted in black: sequences generated in this study. Highlighted in gray: Wildlife Europe 2006-2009 subgroup.
Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2
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Canine distemper virus in Italian dogs
These findings are further supported by the phylogenetic tree, which depicts 6 viruses clustering in the Arctic-like lineage and 741.2006 in the Europe 1/South America 1 lineage (Figure 1). Furthermore, 99.2011, 319.2012 and 352.2012, formed a monophyletic clade inside the Arctic-like lineages, strictly related with a North Italian CDV identified in 2008 (HM443706). The H protein of the 6 Arctic-like CDVs exhibited residues asparagine (N) at position 530 and tyrosine (Y) at position 549, in accordance with all Arctic-like strains sequenced until now; whereas, in accordance with all Europe 1 strains identified in domestic dogs, 741.2006 exhibited residues glycine (G) at position 530 and tyrosine (Y) at position 549 (Martella et al. 2007, Monne et al. 2011). Some differences were detected in other amino acid positions, as shown in Figure 2. In particular, 99.2011, 319.2012 and 352.2012 exhibit methionine (M) at position 445 and serine (S) at position 559 which were never reported previously in other CDVs. Furthermore, these latter Arctic-like viruses exhibit threonine (T) at position 417 and asparagine (N) at position 435 previously reported in another North Italian CDV strain HM443706.
These data show a considerable spread of Arctic‑like lineage in the sampled dogs, in agreement with previous reports on CDV strains collected in Italy which had revealed the presence of Arctic-like CDVs in dogs since 2000 and confirm the dissemination of this novel genotype in Italy (Martella et al. 2006, Martella et al. 2007, Monne et al. 2011). The Arctic‑like lineage is native to the Arctic ecosystem and was usually related to infection of wild animals; it was only occasionally associated with outbreaks in domestic dogs from country geographically distant from Italy, such as Hungary, United States of America, China and Greenland (Blixenkrone‑Möller et al. 1992, Demeter et al. 2007, Pardo et al. 2005). Subsequently to the arrival of Arctic-like CDVs in Italy, probably due to a significant movement of viruses from the East‑Central Europe to Italy in consequence of intense trade of dogs (Martella et al. 2006), the increasing detection of these viruses might suggest that strains belonging to Arctic‑like lineage are becoming progressively endemic in Italy. Furthermore, the 3 viruses more recently identified (99.2011, 319.2012 and 352.2012) showed 4 distinctive amino acid mutations compared to all other Arctic CDVs. Although the study shows a high prevalence of Arctic-like CDV in sampled dogs, the number of viral
Figure 2. Amino acids differences in the sequence tract of H gene between the 7 viral strains identified in dogs in Italy between years 2002-2012. The ruler at the top shows the amino acid positions corresponding to the entire H protein.
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strains analysed should be increased, extending the monitoring activities also to other geographical areas, to properly assess the incidence of Arctic-like CDV strains in Italian dogs. Further studies would
Canine distemper virus in Italian dogs
be necessary to understand what benefits the new amino acid mutations can confer to the Arcticlike CDVs and in which way these may affect their spreading in Italy and in Europe.
References Blixenkrone-Möller M., Svansson V., Appel M., Krogsrud J., Have P. & Orvell C. 1992. Antigenic relationships between field isolates of morbilliviruses from different carnivores. Arch Virol, 123(3-4), 279-294. Calderon M.G., Remorini P., Periolo O., Iglesias M., Mattion N. & La Torre J. 2007. Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina. Vet Microbiol, 125(3-4), 341-349. Demeter Z., Lakatos B., Palade E.A., Kozma T., Forgách P. & Rusvai M. 2007. Genetic diversity of Hungarian canine distemper virus strains. Vet Microbiol, 122(3-4), 258-269. Di Francesco C.E., Di Francesco D., Di Martino B., Speranza R., Santori D., Boari A. & Marsilio F. 2012. Detection by hemi-nested reverse transcription polymerase chain reaction and genetic characterization of wild type strains of Canine distemper virus in suspected infected dogs. J Vet Diagn Invest, 24(1), 107-115. Martella V., Blixenkrone-Møller M., Elia G., Lucente M.S., Cirone F., Decaro N., Nielsen L., Bányai K., Carmichael L.E. & Buonavoglia C. 2011. Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain. Vaccine, 29(6), 1222-1227. Martella V., Cirone F., Elia G., Lorusso E., Decaro N., Campolo M., Desario C., Lucente M.S., Bellacicco A.L., Blixenkrone-Møller M., Carmichael L.E. & Buonavoglia C. 2006. Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy. Vet Microbiol, 116(4), 301-309. Martella V., Elia G., Lucente M.S., Decaro N., Lorusso E., Banyai K., Blixenkrone-Møller M., Lan N.T., Yamaguchi R., Cirone F., Carmichael L.E. & Buonavoglia C. 2007. Genotyping canine distemper virus (CDV) by a hemi-nested multiplex PCR provides a rapid approach for investigation of CDV outbreaks. Vet Microbiol, 122(1-2), 32-42. McCarthy A.J., Shaw M.A. & Goodman S.J. 2007. Pathogen
Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2
evolution and disease emergence in carnivores. Proc Biol Sci, 274(1629), 3165-3174. Monne I., Fusaro A., Valastro V., Citterio C., Dalla Pozza M., Obber F., Trevisiol K., Cova M., De Benedictis P., Bregoli M., Capua I. & Cattoli G. 2011. A distinct CDV genotype causing a major epidemic in Alpine wildlife. Vet Microbiol, 150(1-2), 63-69. Nikolin V.M., Wibbelt G., Michler F.U., Wolf P. & East M.L. 2012. Susceptibility of carnivore hosts to strains of canine distemper virus from distinct genetic lineages. Vet Microbiol, 156(1-2), 45-53. Panzera Y., Calderón M.G., Sarute N., Guasco S., Cardeillac A., Bonilla B., Hernández M., Francia L., Bedó G., La Torre J. & Pérez R. 2012. Evidence of two co-circulating genetic lineages of canine distemper virus in South America. Virus Res, 163(1), 401-494. Pardo I.D., Johnson G.C. & Kleiboeker S.B. 2005. Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America. J Clin Microbiol, 43(10), 5009-5017. Scagliarini A., Dal Pozzo F., Gallina L., Vaccari F. & Morganti L. 2007. TaqMan based real time PCR for the quantification of canine distemper virus. Vet Res Commun, 31(Suppl 1), 261-263. Sekulin K., Hafner-Marx A., Kolodziejek J., Janik D., Schmidt P. & Nowotny N. 2011. Emergence of canine distemper in Bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein. Vet J, 187(3), 399-401. Zhao J.J., Yan X.J., Chai X.L., Martella V., Luo G.L., Zhang H.L., Gao H., Liu Y.X., Bai X., Zhang L., Chen T., Xu L., Zhao C.F., Wang F.X., Shao X.Q., Wu W. & Cheng S.P. 2010. Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes, raccoon dogs and minks in China. Vet Microbiol, 140(1-2), 34-42.
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