Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America

Abstract

In 2004, six puppies and one adult dog from a total of four premises were subjected to necropsy evaluation. For five of the seven dogs, disease caused by canine distemper virus (CDV) infection was suspected based on clinical signs. In all of the dogs, a diagnosis of CDV infection was established by the presence of compatible gross and histologic lesions, immunohistochemical labeling for CDV antigen, and detection of CDV RNA by reverse transcription-PCR. To further characterize the CDV strains detected in the four cases, complete gene sequences were determined for the hemagglutinin (H) and fusion (F) protein genes, while partial gene sequencing was performed for the phosphoprotein gene. A total of 4,508 bases were sequenced for the CDV strains detected from each of the four cases. Two cases were found to have identical sequences except for 2 bases in the intergenic region of the F and H genes. Phylogenetic analysis strongly suggested an evolutionary relationship between sequences detected in these two cases and those of phocine distemper virus 2 and two other strains of CDV not previously detected in the continental United States. Clear phylogenetic relationships were not established for viruses detected in the two additional cases; however, one strain showed similarity to CDV strains detected in a panda from China. Importantly, the three CDV strains detected were demonstrated to be genetically distinct from known vaccine strains and strains previously reported in the continental United States.

FIG. 1.

Alignment of deduced amino acid sequences from hemagglutinin genes of CDV strains. Only amino acids that differ from the majority sequence are shown. Identical residues are shown by dots. Potential N-linked glycosylation sites (N-X-S/T) are shaded. Cysteine (▾) and proline (▪) residues are indicated. The predicted amino acid sequences of case 21261 were identical to those shown for case 18133. Chn, China; Dnk, Denmark.

FIG. 2.

Phylogenetic tree for the complete H gene sequences of representative CDVs plus those detected in cases 18133, 21261, 19876, and 25259 (shown in boxes). The unrooted tree was generated using the distance matrix program NEIGHBOR with the jumble option invoked. Distance values were calculated by the DNADIST program within the PHYLIP software package using the Kimura two parameter. Only bootstrap values greater than 70 are shown, and the branch lengths are proportionate to genetic distances. The country of origin of each CDV is indicated by a two- or three-letter abbreviation following the isolate designation; Chn, China; Jpn, Japan; Ger, Germany; SA, South Africa; USA, United States of America; Dnk, Denmark; Trk, Turkey; Twn, Taiwan; Gnd, Greenland; Sib, Siberia. Canine distemper viruses used for comparison with the country of origin and year of isolation (if known), plus the accession numbers, are as follows: A75/17 (USA, 1975), AF164967; black leopard (USA, 1991), Z47763; black panther, strain A92-6 (USA, 1992), Z54166; Chinese leopard, strain A92-27/4 (USA, 1992), Z54156; dog (USA, 1989), Z47762; dog 4513 (Ger), Z77673; dog (Taiwan), AY378091; dog Hamamatsu (Japan, 1992 to 1994), D85754; dog Yanaka (Japan, 1992 to 1994), D85755; dog 5804 (Germany, 1990), AY386315; dog 91A (Denmark, 1991), AF478544; dog 91B (Denmark, 1991), AF478546; dog 2544 (Germany, 1995), Z77672; dog (Turkey), AY093674; dog (China), AF172411; dog GR88 (Greenland, 1988), Z47760; dog 26D (Japan, 1999), AB040766; dog 5B (Japan, 1999), AY297453; dog 98-002 (Japan, 1998), AB025270; dog HM3 (Japan, 1999), AB040767; dog KDK-1 strain (Japan, 1991), AB025271; dog Ueno (Japan, 1992 to 1994), D85753; ferret (German, 1989), X84999; giant panda (China), AF178038; javelina (USA, 1989), Z47764; lesser panda (China), AF178039; mink (Denmark, 1986), Z47759; Onderstpoort (South Africa), AF378705; PDV-2 Siberian seal (Siberia, 1988), X8499; raccoon 98-2646 (USA, 1988), AY542312 (also represents raccoon 98-2655 [USA, 1998], AY548109, and raccoon 98-2666 [USA, 1998], AY548110); raccoon 01-2689 (USA, 2001), AY465925 (also represents raccoon 01-2690 [USA, 2001]); raccoon 00-2601 (USA, 2000), AY443350; raccoon 01-2641(USA, 2001), AY526496; raccoon 01-2676 (USA, 2001), AY498692; raccoon 98-2654 (USA, 1998), AY466011; raccoon (USA, 1989), Z47765; raccoon dog (Japan, 1996), AB016776; Snyder Hill (Germany), AF259552.

FIG. 3.

Phylogenetic tree for the complete F gene sequences of representative CDVs plus those detected in cases 18133, 21261, 19876, and 25259 (shown in boxes). The unrooted tree was generated using the distance matrix program NEIGHBOR with the jumble option invoked. Distance values were calculated by the DNADIST program within the PHYLIP software package using the Kimura two parameter. Only bootstrap values greater than 70 are shown, and the branch lengths are proportionate to genetic distances. The country of origin of each CDV is indicated by a two- or three-letter abbreviation following the isolate designation: Ger, Germany; SA, South Africa; USA, United States of America; Sib, Siberia. Canine distemper viruses used for comparison with the country of origin and year of isolation (if known), plus the accession numbers, are as follows: A75/17 (USA, 1975), AF164967; dog 5804 (Germany, 1990), AY386315; dog 2544 (Germany, 1995), AJ007711; raccoon 00-2601 (USA, 2000), AY443350; raccoon 01-2689 (USA, 2001), AY649446; raccoon 98-2646 (USA, 1998), AY542312; raccoon 98-2654 (USA, 1998), AY466011; raccoon 98-2645 (USA, 1998), AY445077; Onderstepoort (South Africa), AF014953; PDV-2 (Siberia, 1988), L07075.

FIG. 4.

Phylogenetic tree for partial P gene sequences of representative CDVs plus those detected in cases 18133, 21261, 19876, and 25259 (shown in boxes). The unrooted tree was generated using the distance matrix program NEIGHBOR with the jumble option invoked. Distance values were calculated by the DNADIST program within the PHYLIP software package using the Kimura two parameter. Only bootstrap values greater than 70 are shown, and the branch lengths are proportionate to genetic distances. The country of origin of each CDV is indicated by a two- or three-letter abbreviation following the isolate designation: Bul, Bulgaria; Jpn, Japan; Ger, Germany; SA, South Africa; USA, United States of America; Sib, Siberia; Swd, Sweden. Canine distemper viruses used for comparison with the country of origin and year of isolation (if known), plus the accession numbers, are as follows: A75/17 (USA, 1975), AF164967; dog Alaska (USA, 2003) (25); dog Hamamatsu (Japan, 1992 to 1994), AB028915; dog Jujo (Japan), AB028916; dog Yanaka (Japan, 1992 to 1994), AB028914; dog (Germany, 1993), AF259549; dog 5804 (Germany, 1990), AY386315; ferret (Germany, 1989), AF259550; PDV-2 (Siberia, 1988), AF259551; raccoon 98-2645 (USA, 1998), AY445077; raccoon 98-2646 (USA, 1998), AY542312; raccoon 98-2654 (USA, 1998), AY466011; raccoon 00-2601 (USA, 2000), AY443350; Rockborn (Sweden), AF181446.