Anarysis of vector transmission, genome straucture

植物病毒基因组的分子克隆和测序在过去的十年中已经完成。分子分析的目的之一是改进检测和诊断致病病毒的方法。对于分子分析，通常从纯化的病毒制剂中提取病毒核酸。然而，存在许多不能通过当前方法纯化的寄生虫病毒，因此，病毒核酸样品和它们的抗血清不可用。黄瓜黄化病毒（Cucumber Yellows Virus，CuYV）就是其中之一。本研究应用植物dsRNA提取技术，从植物中提取病毒RNA，复制型dsRNA（RF-dsRNA）。本研究的第一个目的是制备CuYV的cDNA克隆，并测定CuYV基因组的完整核苷酸序列。这是世界上第一个记录，表明CuYV是Crinivirus属的成员。在印度尼西亚也发现了CuYV，但在巴基斯坦没有发现。然而，该病毒不能通过PCR从其粉虱载体中检测到。番茄传染性褪绿病毒是日本新近发现的一种蔓状病毒属病毒。我们还尝试使用dsRNA提取技术来构建质粒以在大肠杆菌中表达病毒外壳蛋白。最后成功地制备了抗胡萝卜潜病毒的抗血清，表明该方法可用于制备目前尚无抗胡萝卜潜病毒血清的抗血清。

Molecular cloning and sequencing of plant virus genomes has been carried out during the past decade. One objective of molecular analysis is improvement of the methods to detect and diagnose pathogenic viruses. For molecular analysis, viral nucleic acids are usually extracted from purified virus preparations. However, there are many recalcitrant viruses that are unable to purify by current methods and, therefore, the viral nucleic acid samples and their antisera are not available. Cucumber yellows virus(CuYV) is one of such recalcitrant viruses. In this research, the application of dsRNA extraction technique from plants was applied to get viral RNA, replicative form of dsRNA(RF-dsRNA). The first objective of this research was the production of cDNA clones of CuYV and complete nucleotide sequence of CuYV genome was determined. This is the first record in the world and suggests that CuYV is a member of the genus Crinivirus. Occurrence of CuYV was also detected in Indonesia but not in Pakistan. However, the virus could not be detected from its whitefly vector by PCR. In this research Tomato infectious chlorosis virus, a member of criniviruses, was newly recognized in Japan. We have also attempted that the dsRNA extraction technique was used to construct a plasmid to express the viral coat protein in E.coli. Finally production of the antiserum against carrot cryptic virus was succeeded, indicating that this method is useful to prepare antisera against ciriniviruses for which no antisera are available in present.