Molecular Identification of a Recent Type of Canine Distemper Virus in Japan by Restriction Fragment Length Polymorphism.

Abstract

ABSTRACT. Restriction fragment length polymorphism analysis was used to differentiate recent field viruses of canine distemper virus (CDV) from vaccine strains. Virus genomes were amplified by using reverse transcriptase-polymerase chain reaction in part of the haemagglutinin gene. After digestion with EcoRV, the PCR products of recent field isolates were cut into two fragments that differ from the uncut form of old strains including all of vaccine strains. This method could be applied to fresh or stored brains, spleens and peripheral blood mononuclear cells of infected dogs. This molecular approach is useful for determining the causative agent of postvaccinated CDV infection. -KEY WORDS: canine distemper, haemagglutinin, RFLP, RT-PCR, vaccine.J. Vet. Med. Sci. 60(11): 1209-1212, 1998 in 1977), DKC6 (Tokyo, 1984), Hamamatsu (Shizuoka, 1993), Yanaka (Tokyo, 1994), Adachi (Tokyo, 1995) [7, 18], Jujo (Tokyo, 1996) and Hamanako (Shizuoka, 1997) strains. The latter two strains were newly isolated from peripheral blood mononuclear cells (PBMCs). The laboratory strains and the MD77 and DKC6 strains were propagated in Vero cells, while the other field isolates were propagated in B95a cells. Experimentally and naturally CDV-infected Dogs:Two specific-pathogen-free beagle puppies were experimentally infected with the Yanaka strain intracerebrally or intranasaly and intravenously, and then euthanized at seven days post inoculation (dpi). Five dogs were diagnosed as CDVinfected by veterinary clinicians. One of them (Dog No. c94037) died from the infection and the other four (c94071, c97006, c97007 and c97023) were euthanized at the moribund stage. Fifty to 100 mg of brains and spleens or PBMCs from these experimentally or naturally infected dogs were used for RNA extraction. PBMCs were separated from heparinized whole blood on Ficoll-paque (Pharmacia LKB, Biotechnology AB, Uppsala, Sweden) [12]. In addition to extracting RNAs from PBMCs, we attempted isolation of CDV as described previously [13]. All of the naturally infected dogs were diagnosed as having CD by clinical signs and histopathological studies.RT-PCR and RFLP analysis: Total RNA was extracted from the virus-infected cells, tissues or PBMCs using ISOGEN (Nippongene, Tokyo, Japan) according to the manufacturer's instructions. RT-PCR was performed using BcaBEST TM RNA PCR Kit (TaKaRa, Siga, Japan). Random 9 mer was used for the RT reaction and two primer sets, CDVH17 and CDVH16, and CDVH15 and CDVHd6 [12], were used for PCR. One µg of total RNA was subjected to the RT reaction according to the manufacturer's instructions, and then to denaturation and extension at 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min for 30 cycles for infected culture cells or for 50 cycles for tissues and PBMCs,

Characterization of a new dog isolate of canine distemper virus from China

“…Since the hydrophobicity of the amino acid sequence of a protein reflects its antigenic determinants (Jameson and Wolf, 1988), the variation of the epitope aa 157-169 may be related to the failure of the protection of the dog after the CDV Onderstepoort vaccination. Surveys conducted in 2 animal hospitals in Tokyo, Japan revealed that 2/3 of vaccinated dogs were diagnosed with CD, and most of them had high neutralization antibody titers against the Onderstepoort strain, but very low titers against wt strains, suggesting that the antigenic epitopes on the envelope of vaccine strain are different from wt strains (Ohashi et al, 1998;Iwatsuki et al, 2000). Moreover, Iwatsuki believed that different N-linked glycosylation sites could affect the immunogenicity of the H protein (Iwatsuki et al, 2000).…”

Update on molecular techniques for diagnostic testing of infectious disease

“…RFLP analysis is used commonly to document the existence of pathogenic versions of bacteria such as Escherichia coli [26]. Manifestations of canine distemper virus or feline panleukopenia virus infections that follow closely on the heels of vaccination can be examined by use of the molecular techniques to determine if the clinical disease is caused by the vaccine isolate or a field isolate [27,28].…”

Detecção do vírus da cinomose canina por RT-PCR utilizando-se oligonucleotídeos para os genes da fosfoproteína, hemaglutinina e neuraminidase

“…Vários relatos, porém, e estudos epidemiológicos revelaram aumento na incidência da infecção por este agente tanto em cães vacinados como em não vacinados (Jozwik e Frymus, 2002). Vários estudos evidenciaram diferenças antigênicas entre amostras vacinais e selvagens (Ohashi et al, 1998;Haas et al, 1999;Hashimoto et al, 2001;Negrão et al, 2006).…”