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Phylogenetic analysis of Austrian canine distemper virus strains from clinical samples from dogs and wild carnivores
V. Benetka, M. Leschnik, N. Affenzeller, K. Mstl
Austrian field cases of canine distemper (14 dogs, one badger [Meles meles] and one stone marten [Martes foina]) from 2002 to 2007 were investigated and the case histories were summarised briefly. Phylogenetic analysis of fusion (F) and haemagglutinin (H) gene sequences revealed different canine distemper virus (CDV) lineages circulating in Austria. The majority of CDV strains detected from 2002 to 2004 were well embedded in the European lineage. One Austrian canine sample detected in 2003, with a high similarity to Hungarian sequences from 2005 to 2006, could be assigned to the Arctic group (phocine distemper virus type 2-like). The two canine sequences from 2007 formed a clearly distinct group flanked by sequences detected previously in China and the USA on an intermediate position between the European wildlife and the Asia-1 cluster. The Austrian wildlife strains (2006 and 2007) could be assigned to the European wildlife group and were most closely related to, yet clearly different from, the 2007 canine samples. To elucidate the epidemiological role of Austrian wildlife in the transmission of the disease to dogs and vice versa, H protein residues related to receptor and host specificity (residues 530 and 549) were analysed. All samples showed the amino acids expected for their host of origin, with the exception of a canine sequence from 2007, which had an intermediate position between wildlife and canine viral strains. In the period investigated, canine strains circulating in Austria could be assigned to four different lineages reflecting both a high diversity and probably different origins of virus introduction to Austria in different years.
CANINE distemper is a widespread, multisystemic, often fatal disease of dogs and other species of Canidae, Mustelidae, Procyonidae, Ursidae and the large Felidae. A similar disease, which affects pinnipeds and is known as phocine distemper, is caused by phocine distemper virus (PDV). Viraemia often leads to the invasion of the CNS, and the outcome in animals with severe clinical CNS signs is nearly always fatal (Greene and Appel 2006). Canine distemper virus (CDV) is a single-stranded RNA virus in the order Mononegavirales, family Paramyxoviridae and genus Morbillivirus. The complete genome consists of 15,690 bases encoding seven proteins (Sidhu and others 1993). The fusion (F) protein is an integral membrane protein binding cell surface receptors of target cells. The F protein and the haemagglutinin (H) protein are both envelope proteins involved in viral-induced cell-to-cell fusion, cytopathogenicity and cell tropism. When there is interchange of H proteins between different strains of CDV and the measles virus, and of both F and H proteins between highly and less pathogenic CDV strains, recombinant strains display the characteristics of the original donator strains, showing higher or lower cytopathogenicity in cell culture and virulence in the animal model than their non-recombinant counterparts (von Messling and others 2001, 2003). Sequence analyses of the variable H genes of field isolates have revealed different lineages of CDV strains circulating worldwide (Bolt and others 1997, Haas and others 1997, Iwatsuki and others 1997, Pardo and others 2005, Martella and others 2006, Demeter and others 2007): lineage USA-1, with the vaccine strains Convac, Onderstepoort, Snyder-Hill; lineage USA-2, with US isolates from different hosts (canine and wildlife) closely related to each other yet distinct from the vaccine strains; the Asian cluster (Asia-1 and Asia-2); and the European cluster, consisting of the European wildlife group and the so-called Arctic lineage formed by sequences similar to PDV-2. A cluster of sequences from the USA and China (AF178038, red panda [Ailurus fulgens]; AF178039, giant panda [Ailuropoda meanoleuca]; and AY964114, dog) occupies an intermediate position between the Asia-1 and the European wildlife group. Their classification into one of these two groups varies between the different genomic regions investigated and the authors who conducted the research (Martella and others 2006, McCarthy and others 2007, An and others 2008, Han and others 2008). Arctic-like sequences have been detected in dogs in Italy and Hungary. Therefore, overall, the clustering of CDV isolates seems to be more related to geographic than host origin (Harder and others 1996, Zhao and others 2010). However,
April 9, 2011 | Veterinary Record

Veterinary Record (2011) 168, 377 V. Benetka, DVM, K. Mstl, DVM, Clinical Virology, Department for Pathobiology, M. Leschnik, DVM, N. Affenzeller, DVM, Clinic of Internal Medicine and Infectious Diseases, Department for Companion Animals and Horses, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria

doi: 10.1136/vr.c6404 Correspondence to Dr Mstl, e-mail: karin.moestl@vetmeduni.ac.at Provenance: not commissioned; externally peer reviewed Published Online First April 6, 2011

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Lednicky and others (2004) described CDV infections in raccoons (Procyon lotor) that differ in virulence and growth characteristics depending on the year of isolation. McCarthy and others (2007) have identified, in particular, two amino acid residues in the H protein associated with host specificity. Substitutions in these two positions (549 and 530) are predominantly related to host switches to non-dog hosts. These are substitutions of glycine (G) or glutamic acid (E) at position 530 in canine samples to aspartic acid (D), asparagine (N) or arginine (R) in non-dog hosts, and substitutions of tyrosine (Y) at position 549 in canine samples to histidine (H) in other hosts. In order to obtain more information about the strains and lineages of CDV circulating in Austria in both dogs and wildlife, F and H gene sequences of selected CDV-positive field samples from the years 2002 to 2007 were amplified using specifically designed PCRs and subsequently submitted for phylogenetic analysis. Amino acid substitutions related to possible cross-species transmission, in particular at positions 549 and 530, were analysed. was carried out in a volume of 20 l with a ready to use sequencing PCR mixture (DNA Sequencing Kit; Applied Biosystems); for each sample, the forward and reverse sequences were determined. Three sections of the viral genome were aligned and used for phylogenetic analyses (bp positions relative to the strain Onderstepoort; GenBank accession number AF378705): F gene segment 1, position 5200-5980, 781 bp; F gene segment 2, position 6284-6923, 640 bp; and H gene segment 1, position 7079-7878, 800 bp. For a selection of available samples (Fig 1), the complete H gene, position 7079-8902, 1824 bp was used. For alignment of nucleotide and amino acid sequences, the program Align Plus 4 (Scientific and Educational Software) and the free ClustalX (www.clustal.org) software were used. Phylogenetic trees were constructed using the program MEGA 4.1 (www.megasoftware. net).

Results
Case histories
Dogs Of the 14 dogs with CDV infection, only two were older than one year, four were eight to 12 months old and eight were six months old or younger (Table 1). All had high-risk origins or circumstances such as exposure in an animal shelter, importation, unknown origins or contact with dogs with suspected CDV infection. The only animal with a complete and documented vaccination history was a male seven-year-old American Staffordshire terrier (3148-03) owned by an Austrian animal shelter. This dog had not been vaccinated for 2.5 years when it was presented at the end of April 2003 with fever, diarrhoea, purulent conjunctivitis, bronchopneumonia, general muscle atrophy, particularly of the masticatory muscles, and myoclonus of the pelvic limbs. Because of the progression of CNS signs, the dog was euthanased. All other dogs were either too young for (complete) vaccination, not vaccinated or had an unknown vaccination history. Clinically, 13 of the 14 dogs showed respiratory signs, 11 had gastroenteritis, eight had hyperkeratosis and 10 had CNS signs. Eleven dogs were euthanased or died, while the three survivors

Materials and methods

Blood samples from 14 dogs, a stone marten (Martes foina) and a badger (Meles meles) were investigated (T able 1).

Primer design and RT-PCR assays

Using primer design software (Scientific & Educational Software, v4.10), seven primer pairs amplifying a total of more than 3.5 kb of the F and H protein genes (Table 2) were designed. RT-PCR assays were run in a volume of 20 l, made up of 18.4 l reaction mixture (OneStep RT-PCR Kit; Qiagen) and 1.6 l template. The cycler scheme consisted of two pre-PCR steps at 50C for 30 minutes and 95C for 15 minutes, followed by 40 cycles at 94C for 30 seconds, 55C and 72C for one minute each, and a final extension of 72C for 10 minutes.

Sequence and phylogenetic analyses

Amplified DNA was extracted using a commercially available kit (NucleoSpin Extract; Machery-Nagel) following the manufacturers instructions, and served as a template for PCR sequencing. The latter

TABLE 1: Cases of canine distemper virus investigated in 14 dogs, a stone marten (Martes foina) and a badger (Meles meles) in Austria during 2002 to 2007
Animal species, sample ID Dog, 4520-02 Dog, 5102-02 Dog, 5253-02 Dog, 5332-02 Dog, 5417-02 Dog, 5428-02 Dog, 5470-02 Dog, 5527-02 Dog, 5825-02 Dog, 3148-03 Dog, 4088-03 Dog, 2779-04 Dog, 2727-07 Dog, 2730-07 Badger, 3156-06 Stone marten, 2390-07 Age 6m 6m 1y 1y 4m 1.5 y 1y 2m 5m 7y 5m 8m 2m 2m Adult Adult Origin or circumstances Imported from Portugal, animal shelter Acquired seven weeks before, origin unknown Imported, origin unknown Animal shelter Imported from Spain, contact animal in Spain died In contact with a CDV-positive dog Animal shelter Imported from Spain, in Austria for four months Stray dog found four weeks before onset of clinical signs Imported from Hungary, all littermates died Three out of a litter of nine alive, dam diseased Abnormal behaviour Vaccination history Unknown No vaccination Unknown No vaccination Single dose No vaccination Unknown No vaccination No vaccination 2.5 years before Single dose Single dose Single dose No vaccination NA NA Clinical signs R+GI R+CNS+H R+GI+CNS+H R+CNS+GI+H R+GI+H R+GI+CNS R+CNS+H R R+GI+CNS+H R+GI+CNS R+GI+CNS+H GI+CNS R+GI+ CNS R+GI+H Unknown CNS? Outcome Euthanased Died Euthanased Euthanased Released to home care Euthanased Euthanased Released to home care Euthanased Euthanased Euthanased Released to home care Euthanased Euthanased Found dead Killed

CDV Canine distemper virus, GI Gastrointestinal, H Hyperkeratosis, m Months, NA Not applicable, R Respiratory, y Years

TABLE 2: Oligonucleotides used for the amplification of F and H genes (annealing temperature 55C) of selected canine distemper virus-positive samples from Austria during 2002 to 2007
Primer CDV 1 CDV 2 CDV 3 CDV 4 CDV 5 CDV 6 CDV 7 Forward primer sequence 5-AGCAAGCCAGCCACAGATCG-3 5-CTCTAATGACCAAGAATGTG-3 5-TGTGTATTCGTCTCAGAATC-3 5-ACGCTACCAACAGACACTCA-3 5-ATCACCAAGTCATAGATGTC-3 5-TGCCATTACTCCAGACAACC-3 5-CGGTCCGGTTATACTGAATG-3 Reverse primer sequence 5-TGCACCTGCAAGCACCACTC-3 5-GCATAACTCAGTGCTTGAAT-3 5-ATTCTGCTTGAGTGTCTGTT-3 5-GAGCGGTGTCAAGACATCTA-3 5-ATAGTTGGTTGTCTGGAGTA-3 5-TCCATACCGTCTCCATTCAG-3 5-CATGCCTAAGGCCAATTGAG-3 Size (bp) 497 436 585 537 560 526 582 Position on strain Onderstepoort (GenBank accession number AF378705) 5137-5633 5545-5980 6270-6854 6827-7363 7332-7891 7866-8391 8359-8940 Gene F gene F gene F gene F and H gene H gene H gene H gene

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AY526496_rac GQ214369_mar GQ214374_bad GQ214371_dog GQ214384_dog GQ214379_dog GQ214383_dog GQ214380_dog GQ214376_dog GQ214378_dog X84998_PDV2. GQ214373_dog 1351 1351 1351 57 71 71 64 1351 1351 1351 1371 1351
TIPPKNGTVLGLINKASRGDQFTV TPHVLTFAPRESSGNCYLPIQTSQIMDKDVLTESNLVVLPTQDFRYVIATYD ISRGDHAI VYYVYDPIRTISYTHPFRLTTKGRPDFLR .. . . . . . . . . . ... . . . . . G . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . D . . .. . . . . . . .. . ... . . . . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . G . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . D . . .. . . . . . . .. . ... . . . . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . F . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . D . . .. . . . . . . .. . K. . F . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . . . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . .. . . . . . . I . . ... . . . . . . . . . . I . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . N . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . . ... . . . . . I . . ... . . . . . . . . . . I . . . .S . . . . . . . . . . . .. . . . . . . . . . . . .. . . . .. . . . . . N . . . ... . . . .. . N . . .. . . . . . . .. . ... . . Y . . . .. . . . . . . . . .

FIG 1: H protein amino acid alignment of selected Austrian canine distemper virus samples and phocine distemper virus type 2 (PDV-2): GQ214369/2390-07/martesfoina/AUT/2007, GQ214473/3156-06/melesmeles/AUT/2006, GQ214371/2730-07/dog/AUT/2007, GQ214384/5825-02/dog/AUT/2002, GQ214379/5332-02/dog/2002, GQ214383/5527-02/dog/2002, GQ214380/5417-02/dog/AUT/2002, GQ214376/4520-02/dog/2002, GQ214378/5253-02/dog/AUT/2002, GQ214373/3148-03/AUT/2003. Positions 530 and 549 are framed. Reference strain: AY526496, raccoon, USA, 1991

were released for home care where follow-up investigations were not possible. Wildlife Both the badger and stone marten showed pathological alterations that were not specifically related to CDV infection, including signs of gastritis.

Discussion
In the years 2002 to 2007, CDV-positive blood samples from 14 dogs, a badger and a stone marten were available for investigation. T welve of 14 dogs were one year old or younger and, with one exception, either not or incompletely vaccinated. The only animal with a complete and documented vaccination history, but with the last booster administered 2.5 years earlier, was a seven-year-old shelter dog (3148-03). In a high-risk environment like an animal shelter, recommended booster intervals of three years have to be considered critically (Schoder and others 2006). A high percentage of the randomly chosen canine cases showed CNS involvement (71 per cent), explaining the high fatality (of at least 78 per cent, not including the outcome of dogs released to home care). CDV sequences detected in Austria showed homologies of 93 to 100 per cent to each other and clustered in different previously described lineages (Haas and others 1997, Martella and others 2006, Demeter and others 2007). This grouping was similar in all three genomic regions investigated; only dog 3148-03 clustered in the European lineage in the 5-end of the F gene, but in the Arctic group in the 3-end of the F gene and in the H gene. This might reflect a possible recombination event. Circulating CDV strains were largely similar, both in neighbouring geographic regions and in the year of detection. All Austrian CDV H gene sequences detected in 2002 formed a group well embedded in the European lineage, closely related to sequences from Italy in 2002 and 2003 and Hungary in 2004, and also to sequences detected more than a decade earlier in Germany and Denmark. One sequence of this cluster detected in 2004 (2779-04) was nearly identical to a Hungarian isolate from the same year. The Austrian Arctic sequence of 2003 showed the highest identities to Hungarian isolates of 2005 to 2006 and might have been the source of the Hungarian outbreak. Interestingly, both the Hungarian and the Austrian Arctic sequences originated from dogs kept in animal shelters. The two sequences from 2007 (2727-07 and 2730-07) occupy a clearly separate position together with isolates from China (panda, before 2000) and the USA (dog, 2004), which have been assigned to both the European wildlife and the Asia-1 cluster by different authors (McCarthy and others 2007, An and others 2008, Han and others 2008). The wildlife CDV sequences of 2006 and 2007 were most closely related to a sequence from a Hungarian dog detected in 2005 (accession number DQ889189) and to a mink sequence from Denmark in 1986 (accession number Z47759). Although the badger and stone marten were found nearly a year apart, the Morbillivirus detected was very similar in both cases, but clearly distinct from those detected in dogs. Nevertheless, compared with other Austrian sequences, the wildlife sequences of 2006 and 2007 showed the highest identities (95 to 97 per cent) with the canine samples from 2007. Amino acid sequences for both wildlife samples showed substitutions in the two residues of the H protein predominantly related to host switches and possible changes in virulence (530G/E to 530D and 549Y to 549H) (McCarthy and others 2007, Sekulin and others 2011). As expected in CDV sequences, all Austrian canine samples had tyrosine (Y) at position 549. One sample from 2007 (2730-07) occupied
April 9, 2011 | Veterinary Record

Sequence analysis
By sequence analysis, the lowest homologies of Austrian samples were found with the USA-1 cluster, which comprises different old vaccine strains. Compared with the typical representative, strain Onderstepoort, identities varied from 91 to 94 per cent in segment 1 of the F gene, from 93 to 95 per cent in segment 2 of the F gene and from 92 to 93 per cent in the H gene. The sequences obtained from dog samples in 2007 showed higher levels of identity to strain Onderstepoort than the ones obtained in 2002. Austrian CDV sequences showed homologies to each other of between 94 and 100 per cent in the F gene, with a higher diversity in segment 2 (94 to 100 per cent) than in segment 1 (97 to 100 per cent), and of 94 to 100 per cent in the H gene. The wildlife sequences of 2006 and 2007 were 98 to 99 per cent identical to each other and more homologous to the canine samples of 2007 (96 to 97 per cent) than to those obtained in 2002 (93 to 96 per cent). One sequence (3148-03) showed identities of 96 to 97 per cent in the F gene with PDV type 2 (PDV-2) and of 99 per cent in the H gene with several Hungarian Arctic sequences detected in 2005 to 2006.

Phylogenetic analysis
By phylogenetic analysis, the samples showed similar clustering in the three genomic regions investigated, with one exception, 3148-03, which groups in the European lineage in segment 1 (position 5200-5980) of the F gene, but in the Arctic group in segment 2 (position 6284-6923) of the F gene (data not shown) and in the H gene (Fig 2). All Austrian CDV sequences detected in 2002 formed a very closely related group with one 2003 sequence, all well embedded in the European lineage. The Austrian Arctic sequence showed the highest identity with Hungarian isolates. The two canine sequences from 2007 (2727-07 and 2730-07) occupy a clearly separate position in the European wildlife group/Asia-1 group together with AF178038 (red panda, China), AF178039 (giant panda, China) and AY964114 (dog, USA). The two Austrian wildlife sequences (3156-06 and 2390-07), which cluster with sequences from a mink (accession number Z47759, Denmark 1986) and a dog (DQ889189, Hungary 2005), are more closely related to the latter than to any other Austrian sequence.

Analysis of amino acid positions 530 and 549 of the H protein

The two wildlife amino acid sequences (2006 and 2007) have a substitution of 549H; all Austrian canine samples (2002, 2003, 2005 and 2007) have Y at this position, as described for the majority of canine samples worldwide. The wildlife sequences and one canine sample of 2007 (2730-07) have substitutions of 530G/E to 530D, and the Austrian Arctic sequence (3148-03) has a substitution at the same position to 530N (Fig 1).

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FIG 2: Evolutionary relationship of 83 taxa (800 bp of the H gene of canine distemper virus, position 7079-7878). For each GenBank sequence, the following information is given: the GenBank accession number and, if indicated, the strain, host, country of origin and year of detection. Austrian sequences are given in bold (accession number/specimen voucher/species/ year of detection). The evolutionary history was inferred using the neighbour-joining method. The optimal tree with the sum of branch length = 0.66610640 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4 (Saitou and Nei 1987, Tamura and others 2004, 2007)

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rd/USA/199 A Z54156/chinese-leopa 5/US 75-1 000 ain A /USA/2 g/str ccoon 7/do 350/ta 6496 89 AY443 AF1 /19 SA a/U 01 elin 20 /jav A/ 764 US 9 Z47 n/ 98 oo A/1 cc /US /ra on 96 cco /ra 64 65 52 77 9 AY Z4 98 /1 SA /U og /d 62 77 Z4

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al/ /se 98 49 X8 88 /19 nd nla ree HN g/C g/G /do /do 411 60 72 77 Z4 AF1 05 N/20 x/CH 2/fo 5 450 4 EF4 /200 /ITA /dog /2004 087 g/USA 26 108/do DQ2 AY964

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DQ889185/dog/HUN/2006 DQ889184/dog/HUN/2006 DQ889180/dog/HUN/2005 DQ889181/dog/HUN/2005 DQ889179/dog/HUN/2005 DQ889183/dog/HUN/2005 DQ889182/dog/HUN/2005 DQ889178/dog/HUN/2005

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Asia-2

USA-1

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an intermediate position between wildlife and canine sequences. In this sample, there was a substitution at position 530, where aspartic acid (D) replaced glycine (G) or glutamic acid (E). This substitution was also present in both wildlife samples from 2006 and 2007. These findings might be explained by a previous interspecies transmission between wildlife and dogs followed by viral evolution. The Austrian Arctic sequence shows asparagine (N) at position 530, as do several other Arctic-like sequences. In conclusion, as described for other countries, different distinct lineages of CDV sequences were present in Austria in the years 2002 to 2007, with a strong geographic and temporal clustering. Four lineages were found in Austria: the canine samples of 2002 to 2005 were assignable to the European lineage, one canine sample from 2003 belonged to the Arctic lineage, the European wildlife lineage was represented by two wildlife samples from 2006 and 2007, and a fourth cluster was formed by two canine samples from 2007. Amino acid substitutions related to host specificity were present in the two Austrian wildlife samples at both positions of the H protein (530 and 549) known to be predominantly related to host switching. Of the canine samples analysed, both the Arctic sample (3148-03) and one sample from 2007 showed substitutions at position 530 but not at position 549. All sequences have been deposited at GenBank (www.ncbi. nlm.nih.gov), with the accession numbers: GQ214337-GQ214352 (F gene, position 5200-5980 of strain Onderstepoort), GQ214353GQ214355 and GQ214357-GQ214368 (F gene, position 6284-6923) and GQ214369-GQ214384 (H gene, position 7079-7878 or position 7079-8902).
HAN, G. Z., LIU, X. P. & LI, S. S. (2008) Cross-species recombination in the haemagglutinin gene of canine distemper virus. Virus Research 136, 198-201 HARDER, T. C., KENTER, M., VOS, H., SIEBELINK, K., HUISMAN, W., VAN AMERONGEN, G., ORVELL, C., BARRETT, T., APPEL, M. J. & OSTERHAUS, A. D. (1996) Canine distemper virus from diseased large felids: biological properties and phylogenetic relationships. Journal of General Virology 77, 397-405 IWATSUKI, K., MIYASHITA, N., YOSHIDA, E., GEMMA, T., SHIN, Y. S., MORI, T., HIRAYAMA, N., KAI, C. & MIKAMI, T. (1997) Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs. Journal of General Virology 78, 373-380 LEDNICKY, J. A., DUBACH, J., KINSEL, M. J., MEEHAN, T. P., BOCCHETTA, M., HUNGERFORD, L. L., SARICH, N. A., WITECKI, K. E., BRAID, M. D., PEDRAK, C. & HOUDE, C. M. (2004) Genetically distant American canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the USA. Virology Journal 1, 2 MCCARTHY, A. J., SHAW, M. A. & GOODMAN, S. J. (2007) Pathogen evolution and disease emergence in carnivores. Proceedings of the Royal Society B: Biological Sciences 274, 3165-3174 MARTELLA, V., CIRONE, F., ELIA, G., LORUSSO, E., DECARO, N., CAMPOLO, M., DESARIO, C., LUCENTE, M. S., BELLACICCO, A. L., BLIXENKRONEMLLER, M., CARMICHAEL, L. E. & BUONAVOGLIA, C. (2006) Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy. Veterinary Microbiology 116, 301-309 PARDO, I. D., JOHNSON, G. C. & KLEIBOEKER, S. B. (2005) Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America. Journal of Clinical Microbiology 43, 5009-5017 SAITOU, N. & NEI, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406-425 SCHODER, D., BENETKA, V., SOMMERFELD-STUR, I., KOPF, N., WEISSENBACHER, E., PALLAN, C., WALK, K. & MSTL, K. (2006) Untersuchungen zum Antikrper-Status gegen Hundestaupe-Virus und Canines Parvovirus-2 bei Hunden in Niedersterreich und Wien nach unterschiedlichen Impfintervallen. Veterinary Medicine Austria 93, 176-182 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. Veterinary Journal 187, 399-401 SIDHU, M. S., HUSAR, W., COOK, S. D., DOWLING, P. C. & UDEM, S. A. (1993) Canine distemper terminal and intergenic non-protein coding nucleotide sequences: completion of the entire CDV genome sequence. Virology 193, 66-72 TAMURA, K., DUDLEY, J., NEI, M. & KUMAR, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596-1599 TAMURA, K., NEI, M. & KUMAR, S. (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America 101, 11030-11035 VON MESSLING, V., SPRINGFELD, C., DEVAUX, P. & CATTANEO, R. (2003) A ferret model of canine distemper virus virulence and immunosuppression. Journal of Virology 77, 12579-12591 VON MESSLING, V., ZIMMER, G., HERRLER, G., HAAS, L. & CATTANEO, R. (2001) The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. Journal of Virology 75, 6418-6427 ZHAO, J. J., YAN, X. J., CHAI, X. L., MARTELLA, V., LUO, G. L., ZHANG, H. L. & OTHERS (2010) Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes, raccoon dogs and minks in China. Veterinary Microbiology 140, 34-42

Acknowledgements
The authors thank R. Skerlak for medical care of the study animals.

References
AN, D. J., YOON, S. H., PARK, J. Y., NO, I. S. & PARK, B. K. (2008) Phylogenetic characterization of canine distemper virus isolates from naturally infected dogs and a marten in Korea. Veterinary Microbiology 132, 389-395 BOLT, G., JENSEN, T. D., GOTTSCHALCK, E., ARCTANDER, P., APPEL, M. J., BUCKLAND, R. & BLIXENKRONE-MLLER, M. (1997) Genetic diversity of the attachment (H) protein gene of current field isolates of canine distemper virus. Journal of General Virology 78, 367-372 DEMETER, Z., LAKATOS, B., PALADE, E. A., KOZMA, T., FORGCH, P. & RUSVAI, M. (2007) Genetic diversity of Hungarian canine distemper virus strains. Veterinary Microbiology 122, 258-269 GREENE, C. E. & APPEL, M. J. (2006) Canine distemper. In Infectious Diseases of the Dog and Cat. 3rd edn. Ed C. E. Greene. Saunders Elsevier. pp 25-41 HAAS, L., MARTENS, W., GREISER-WILKE, I., MAMAEV, L., BUTINA, T., MAACK, D. & BARRETT, T. (1997) Analysis of the haemagglutinin gene of current wild-type canine distemper virus isolates from Germany. Virus Research 48, 165-171

April 9, 2011 | Veterinary Record

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Phylogenetic analysis of Austrian canine distemper virus strains from clinical samples from dogs and wild carnivores
V. Benetka, M. Leschnik, N. Affenzeller, et al. Veterinary Record 2011 168: 377 originally published online April 6, 2011

doi: 10.1136/vr.c6404

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