Molecular characterization and genotypic lineages of canine distemper virus isolates in Thailand

Abstract

Canine distemper virus (CDV) has been known causing multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used for controlling the disease for many decades, yet a number of CDV infections in vaccinated dog were still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phospoprotein (P), hemagglutinin (H) and fusion protein (F) gene that play an important role in viral pathogenesis and to develop the restriction fragment length polymorphism (RFLP) techniques for effective differentiation among individual wild-type and vaccine lineages. Four commercial vaccine products, 23 conjunctival swabs and 13 necropsied tissue from dog and civets were included in the study. Routinely histopathological study of various organs and immunohistochemistry (IHC) staining on brain tissues were performed. Viral isolation was done in Vero-DST cell line for virological study. Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions of specimens and vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other lineages in Genbank. Typical microscopic lesions of CDV were found in various organs. IHC of brain tissues indicated the characteristic cell tropism upon different CDV lineages. Phylogenetic analysis revealed that CDV field isolates were not related to vaccine lineage and could be divided into two clusters; one belonged to Asia-1 lineage and another, not related to any previous recognized lineages was proposed as ‘new Asia lineage’. RFLP pattern concordantly to phylogenetic trees was able to differentiate among Asia-1, new Asia and vaccine lineage. Regarding previous CDV studies, there were at least 3 lineages of CDV as mentioned circulating in Thailand. Thus, RFLP technique is able to distinguish individual wild-type from vaccine lineage effectively and this method would be useful for several clinical applications in Thailand.