Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells
Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells
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DOI：
10.1128/jvi.77.18.9943-9950.2003
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发表时间：
2003-09-01
影响因子：
5.4
通讯作者：
Yanagi, Y
中科院分区：
文献类型：
--
作者：
Seki, F;Ono, N;Yanagi, Y
我们之前已经证明，犬信号淋巴细胞激活分子（SLAM；也称为 CD150）充当犬瘟热病毒（CDV）的细胞受体。在本研究中，我们建立了稳定表达犬SLAM的Vero细胞（Vero.DogSLAMtag细胞）。接种七只犬瘟热狗中的五只的脾脏样本一天后，在 Vero.DogSLAMtag 细胞中分离出病毒。相比之下，据报道敏感的狨猴 B95a 细胞仅在接种后 7 至 10 天从三只患病动物中成功分离出病毒，并且当使用 Vero 细胞分离时，没有从任何狗身上回收到病毒。在 Vero.DogSLAMtag 细胞中分离的 CDV 毒株不会在 B95a 和表达人类 SLAM 的 Vero 细胞中引起细胞病变效应，而在来自同一狗的 B95a 细胞中分离的毒株却在犬或人类表达 SLAM 的 Vero 细胞以及 B95a 细胞中引起细胞病变效应。这些菌株之间的血凝素序列有两个氨基酸差异。包膜蛋白表达后的细胞融合分析和水疱性口炎病毒假型测定表明，它们的血凝素是造成它们之间细胞趋向性差异的原因。定点诱变表明，为了适应狨猴 SLAM 的使用，需要将血凝素 530 位的谷氨酸替换为赖氨酸。我们的结果表明，稳定表达犬SLAM的Vero细胞对临床标本中的CDV高度敏感，并且仅血凝素中的单个氨基酸取代即可使病毒适应狨猴SLAM。
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.