Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan

Abstract

Canine distemper of domestic dogs is caused by canine distemper virus (CDV), a member of the morbilliviruses. It has been a highly contagious disease of great veterinary importance for centuries, but for the last several decades it has been controlled satisfactorily by modified live vaccines. In the 1990s, however, it was described that CDV strains genetically different from vaccine strains may have caused the disease in vaccinated dogs. The highest antigenic variation is found in the H protein. Therefore, in the present study, hemagglutinin (H) genes obtained from current vaccines and field isolates and amplified directly from clinical specimens were genetically analyzed by restriction fragment length polymorphism assay and sequencing. Phylogenetic analysis of the H-gene amino acid sequences revealed that at least two CDV genotypes are circulating among dogs in Japan; one is a genotype to which almost all Japanese CDV isolates belong and the other has not been previously described. Both are separate and independent from the other lineages or genotypes of vaccine strains, as well as European and U.S. CDV isolates. The results suggest that CDV has also evolved in Japan, and further studies will be needed for an evaluation and possible improvement of the efficacies of current CDV vaccines.

FIG. 1

Outline of the RT-PCR and scheme for RFLP analysis of the CDV H gene. (a) Genomic organization of the CDV genome, the set of primers, and the lengths of the products of the RT-PCRs. The various CDV genes are represented by boxes. The genes for nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin protein (H), and the large protein (L) are indicated. (b) Scheme for the restriction enzyme patterns of CDV H genes digested with FbaI and NdeI. Strains Onderstepoort and KDK-1 were used as representative strains of old and new CDVs, respectively. For strain KDK-1, the third restriction site, which was obtained by digestion with NdeI and which generated the smallest fragment (approximately 100 bp), cannot be specified because no sequence information is available for either end of a PCR product for primers CDV-F8 and CDV-R8.

FIG. 2

Restriction enzyme patterns of CDV H genes digested with FbaI (a) and NdeI (b). Lane M, 100-bp ladder size marker; lanes 1 to 5, field isolates KDK-1, C710D, C714D, C717D, and C720D, respectively; lanes 6 to 10, clinical samples 98/001, 98/003, 98/005, 98-001, and 98-002, respectively; lanes 11 to 17, reference strains Onderstepoort, Fromm, DFE-HC, Lederle VR-128, Rockborn, and FXNO and wild-type strain Snyder Hill, respectively.

FIG. 3

Phylogenetic analysis of the amino acid sequences of the coding regions of CDV H proteins. The tree was constructed by using Clustal W, version 1.74 (32), and PHYLIP, version 3.5 (11), modules. Tree topology was based on the neighbor-joining method. The bootstrap values indicate the number of times that each branching was found in 1,000 bootstrap analyses. Branch lengths indicate phylogenetic distances calculated from distance martices of deduced amino acid sequences. Sequences of CDV strain KDK-1 (accession no. AB02527) and the strain from sample 98-002 (accession no. AB025270) were generated in this study. Other sequences were extracted from the database for the following strains (nucleotide sequence accession numbers are given in parentheses): vaccine strains, Onderstepoort (AF014953, D00758) and Convac (Z35493); U.S. isolates, Chinese leopard/A92-27/4 (Z54156), Black leopard/A92-6 (Z54166), dog/US89 (Z47762), Leopard/US91 (Z47763), Raccoon/US89 (Z47765), Javelina/US89 (Z47765); European isolates, Dog/DK91,B+C (Z47761), Mink/DK86 (Z47759), Dog/404 (Z77671), Dog/2544 (Z77672), Dog/4513 (77673), Ferret/1493/Han89 (X84999), Dog/5804/Han90 (X85000); Asian isolate, Siberian seal/PDV-2 (X84998); Greenland isolate, Dog/GR88 (Z47760); isolates of Japanese origin, Dog/Ueno (D85753), Dog/Hamamatsu (D85754), Dog/Yanaka (D85755), and Racoon dog/Tanu96 (AB016776). The data for the Snyder Hill strain were from reference .