Anarysis of vector transmission, genome straucture

在过去的十年中，对植物病毒基因组进行了分子克隆和测序。分子分析的目标之一是改进检测和诊断致病病毒的方法。对于分子分析，病毒核酸通常从纯化的病毒制剂中提取。然而，有许多顽固的病毒无法用现有的方法进行纯化，因此，无法获得病毒核酸样本及其抗血清。黄瓜黄化病毒(CuYV)就是其中一种顽抗病毒。本研究应用植物dsRNA提取技术获得病毒RNA，即复制形式的dsRNA(RF-dsRNA)。本研究的第一个目标是获得CuYV的cDNA克隆，并测定CuYV基因组的全序列。这是世界上第一个记录，表明CuYV是CriniVirus属的成员。印度尼西亚也发现了CuYV，但巴基斯坦没有发现。然而，从其烟粉虱载体中未检测到该病毒。在本研究中，番茄传染性黄化病病毒是日本新发现的一种病毒。我们还尝试了利用dsRNA提取技术构建在大肠杆菌中表达病毒外壳蛋白的载体。最后成功地制备了胡萝卜潜伏病毒抗血清，表明该方法可用于制备目前尚无抗血清的胡萝卜环状病毒抗血清。

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.