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 CDV

Balboni A

Battilani M.

De Lorenzo Dandola G

Prosperi S

Scagliarini A

English

BALBONI, ANDREA

SCAGLIARINI, ALESSANDRA

PROSPERI, SANTINO

BATTILANI, MARA

Archivio istituzionale della ricerca - Alma Mater Studiorum Università di Bologna

227SHORT COMMUNICATIONDepartment 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: a.balboni@unibo.it.Andrea Balboni*, Giorgia De Lorenzo Dandola, Alessandra Scagliarini,Santino Prosperi & Mara BattilaniCurrently, 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 Presenza di diversi lineage di Canine distemper virus in cani in ItaliaParole chiaveArctic-like CDV,Cane,Canine distemper virus (CDV),Europe 1 CDV,Gene H,Italia.RiassuntoL’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.KeywordsArctic-like,Canine distemper virus,Dog,Europe 1,Gene H,Italy.SummaryThis 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.Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2Accepted: 04.09.2014  |  Available on line: 30.09.2014Occurrence of different Canine distemper virus lineages in Italian dogs228 Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2last 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: KF184985- KF184991.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.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 Canine distemper virus in Italian dogs  Balboni et al.1  www.ncbi.nlm.nih.gov/genbank/. Table I. Details of 7 CDV strains identified in dogs in Italy between years 2002-2012.Virus Sampling date Breed Sex Age Origin Vaccination status Symptoms444 2002 Labrador retriever M 2m Emilia-Romagna Yes R + N456 2003 German shepard F 2m Emilia-Romagna No R + N64 2004 Cane Corso F 1y 3m Emilia-Romagna Yes GI + N + C 741 2006 Mixed-breed Un Un Emilia-Romagna Un Un99 2011 Mixed-breed F 5m Lazio Un GI + N319 2012 Mixed-breed M 2m Lazio Un GI + N352 2012 Mixed-breed F 2m Lazio No NM = male;   F = female;   m = months;   y = years;   R = respiratory symptoms;   N = neurological symptoms;   GI = gastrointestinal symptoms;   C = cutaneous lesions;   Un = unknown.229Balboni et al.  Canine distemper virus in Italian dogs Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2DE_badger_2008_FJ416338DE_fox_2008_FJ416339DE_fox_2008_FJ416336DE_fox_2008_FJ416337IT_badger_2008_HM443704CH_lynx_2010_JF810109IT_fox_2007_HM443705CH_fox_2010_JF810107CH_dog_2010_JF810111IT_fox_2009_HM120874CH_marten_2002_JF810110CH_fox_2010_JF810108CH_fox_2010_JF810106IT_fox_2006_HM443726IT_badger_2006_HM443725IT_fox_2009_HM443709IT_marten_2009_HM443708IT_fox_2008_HM443707HU_dog_2004_DQ889177DE_fox_2007_JN153024DE_fox_2007_JN153025ES_lynx_2005_GU001863ES_marten_2005_GU001864PT_wolf_1998_HM563057PT_wolf_2008_HM563058PT_dog_2007_HM563059AU_dog_2002_GQ214376AU_dog_2002_GQ214378AU_dog_2002_GQ214380AU_dog_2002_GQ214384IT_dog_2003_DQ494319IT_dog_741.2006_KF184988 IT_dog_2002_DQ494318IT_dog_2007_HM443723DK_dog_1994_Z47761IT_dog_2000_HM443718TR_dog_2002_AY093674IT_dog_2003_DQ494317DE_dog_1996_Z77671DE_dog_1996_Z77672DE_dog_1996_Z77673AR_dog_2003_FJ392652UY_dog_2008_JN215474UY_dog_2007_JN215473UY_dog_2008_JN215475UY_dog_2009_JN215476UY_dog_2009_JN215477US_raccoon_2001_AY498692US_dog_1995_Z47762US_raccoon_2001_AY526496US_bleopard_1992_Z47763CN_gpanda_1999_AF178038JP_dog_2005_AB212965JP_dog_1996_D85754JP_badger_2007_AB329581CN_dog_2008_FJ409464CN_fox_2006_EU325722CN_fox_2006_HQ540293AR_dog_2003_AM422846AR_dog_2003_AM422847AR_dog_2005_FJ392651US_dog_2004_AY964110Rockborn-Candur_GU266280US_dog_2004_AY964114Rockborn-46th_GU810819CN_lesser-panda_1999_AF178039IT_fox_2000_DQ228166DK_mink_1992_Z47759HU_dog_2006_DQ889188HU_dog_2006_DQ889189HU_dog_2006_DQ889187AU_badger_2006_GQ214374AU_marten_2007_GQ214369DE_raccoon_2007_JN153022DE_raccoon_2007_JN153019DE_raccoon_2007_JN153020DE_raccoon_2007_JN153021DE_raccoon_2007_JN153023ZA_dog_2007_FJ461714ZA_dog_2007_FJ461698ZA_dog_2007_FJ461720ZA_dog_2007_FJ461724GL_dog_1992_Z47760IT_dog_2000_HM443710IT_dog_2000_HM443713IT_dog_444.2002_KF184985 IT_dog_2000_HM443720IT_dog_2000_HM443719IT_dog_2001_HM443711IT_dog_2000_HM443716IT_dog_2000_HM443717IT_dog_2000_HM443714US_dog_2004_AY964112US_dog_2004_AY964108IT_dog_2005_HM443712IT_dog_2005_DQ226088IT_dog_99.2011_KF184989 IT_dog_319.2012_KF184990 IT_dog_352.2012_KF184991 IT_dog_2008_HM443706IT_dog_2002_HM443721IT_dog_2004_HM443715IT_dog_2004_DQ226087IT_dog_64.2004_KF184987 IT_dog_456.2003_KF184986 IT_dog_2002_HM443724AU_dog_2003_GQ214373IT_dog_2002_HM443722HU_dog_2005_DQ889181HU_dog_2005_DQ889178HU_dog_2005_DQ889179HU_dog_2005_DQ889180HU_dog_2005_DQ889182HU_dog_2005_DQ889183HU_dog_2006_DQ889184HU_dog_2006_DQ889185HU_dog_2006_DQ889186CN_fox_2005_EU743935JP_dog_1998_AB025270JP_dog_2006_AB252718Onderstepoort_AF305419Convac_Z35493Lederle_EF418782SnyderHill_AF259552US_raccoon_1998_AY548109PDV-1_AF479277999199971009810010099991009810098979799869199899597 879388840.05Europe 1South America Proposed LineageAmerica IIAsia ISouth America Proposed LineageRockborn-like Proposed LineageEurope WildlifeSouth AfricaArctic-likeAsia IIAmerica IAFigure 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.230Canine distemper virus in Italian dogs  Balboni et al.Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2These 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 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.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.231Balboni et al.  Canine distemper virus in Italian dogs Veterinaria Italiana 2014, 50 (3), 227-231. doi: 10.12834/VetIt.52.2173.2be necessary to understand what benefits the new amino acid mutations can confer to the Arctic-like CDVs and in which way these may affect their spreading in Italy and in Europe.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. 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Occurrence of different Canine distemper virus lineages in Italian dogs

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

Andrea Balboni

Giorgia De Lorenzo Dandola

Alessandra Scagliarini

Santino Prosperi

Mara Battilani

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Short communication. Occurrence of different Canine distemper virus lineages in Italian dogs

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Occurrence of different Canine distemper virus lineages in Italian dogs

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