Efficient Isolation of Wild Strains of Canine Distemper Virus in Vero Cells Expressing Canine SLAM (CD150) and Their Adaptability to Marmoset B95a Cells

Abstract

We have previously shown that canine signaling lymphocyte activation molecule (SLAM; also known as CD150) acts as a cellular receptor for canine distemper virus (CDV). In this study, we established Vero cells stably expressing canine SLAM (Vero.DogSLAMtag cells). Viruses were isolated in Vero.DogSLAMtag cells one day after inoculation with spleen samples from five out of seven dogs with distemper. By contrast, virus isolation with reportedly sensitive marmoset B95a cells was only successful from three diseased animals at 7 to 10 days after inoculation, and no virus was recovered from any dogs when Vero cells were used for isolation. The CDV strain isolated in Vero.DogSLAMtag cells did not cause cytopathic effects in B95a and human SLAM-expressing Vero cells, whereas the strain isolated in B95a cells from the same dog did so in canine or human SLAM-expressing Vero cells as well as B95a cells. There were two amino acid differences in the hemagglutinin sequence between these strains. Cell fusion analysis after expression of envelope proteins and vesicular stomatitis virus pseudotype assay showed that their hemagglutinins were responsible for the difference in cell tropism between them. Site-directed mutagenesis indicated that glutamic acid to lysine substitution at position 530 of the hemagglutinin was required for the adaptation to the usage of marmoset SLAM. Our results indicate that Vero cells stably expressing canine SLAM are highly sensitive to CDV in clinical specimens and that only a single amino acid substitution in the hemagglutinin can allow the virus to adapt to marmoset SLAM.

Canine Distemper Virus–associated Encephalitis in Free-Living Lynx (Lynx Canadensis) and Bobcats (Lynx Rufus) of Eastern Canada

“…For virus isolation, 10% organ homogenates in Dubelcco's Minimum Essential Medium (DMEM) supplemented with antibiotics were made of lung and brain samples using a tissue homogenizer (Kinematica Polytron, Lucerne, Switzerland). Homogenates were inoculated on Vero.DogSLAM cells for 1 hr at 37 C (Seki et al, 2003). Cell cultures were washed twice with culture medium, DMEM supplemented with antibiotics, and 10% Fetal Calf serum and incubated at 37 C humid atmosphere 5% CO 2 .…”

Lymphotropism and host responses during acute wild-type canine distemper virus infections in a highly susceptible natural host

“…PCR amplification with a primer pair for the cellular glyceraldehyde-3-phosphate dehydrogenase gene, yielding a 568 bp product, served as a control for the RNA extractions (Dahl et al, 2004). The sensitivity of the RT-PCR assay for the Snyder Hill and DK91 wild-type viruses was determined in virusinfected Vero cells expressing dog SLAM (Vero/dSLAM cells) (Seki et al, 2003). The limit of detection of the two-step RT-PCR was in the range 0.03-0.10 TCID 50 in 1 mg total RNA.…”

Growth Characteristics of Canine Distemper Virus in a New Cell Line CCT Cells Originated from Canine Malignant Histiocytosis

“…B95a cell line which is susceptible to CDV as well as MV also expresses abundant SLAM, suggesting SLAM might play as a receptor for CDV [19]. Whereas, dog SLAM has been known to be a real receptor for CDV and we reported an efficient isolation of CDV in Vero cells expressing canine SLAM [16]. However Vero cells were from monkey kidney.…”