Studies on replication, pathogenicity and transmission of plant RNA viruses in the genus Furovirus

土壤传播的小麦花叶病毒（SBWMV）是糠秕病毒属的典型种，其双部正义RNA基因组被封装在棒状病毒粒子中。SBWMV感染小麦和大麦植株，引起花叶病和矮秆病。SBWMV由土栖真菌传播并滞留在土栖真菌中，因此很难从侵染地控制该病毒病。在本研究中，我们对SBWMV的复制、致病性和传播进行了研究，目的是控制该病。构建了日本、美国和英国三株SBWMV的全长感染性cDNA克隆。此外，还对SBWMV进行了侵染试验，并建立了大麦叶肉原生质体系统。利用大麦原生质体对3个分离株进行重配实验，结果表明，RNA1和RNA2组合均支持病毒RNA复制，表明3个地理分离株之间存在密切关系。小麦侵染SBWMV需要低于20℃的温度。用感染性体外转录本接种大麦原生质体，在15 ~ 25℃的温度下进行培养，结果表明，RNA复制的最佳温度为17℃。SBWMV野生型RNA2在17℃传代时频繁发生缺失突变，缺失突变对小麦植株的影响比野生型更为严重。利用接种物的全长感染性RNA2转录本分析缺失突变，发现缺失突变发生在上游衣壳蛋白基因的框架内。症状严重程度与缺失长度相关。为进一步深入分析绒状病毒的复制、致病性和传播特性，本文获得了基本信息、材料和方法。

Soil-borne wheat mosaic virus (SBWMV) is the type species in the genus Furovirus with bipartite plus-sense RNA genome encapsidated in rod-shaped virions. SBWMV infects wheat and barley plants, causing mosaic and stunt diseaese. SBWMV is transmitted by and retained in soil-inhabiting fungus and therefore it is quite difficult to control this virus disease from the infested field. In this study, we investigated replication, pathogenicity and transmission of SBWMV for the purpose of controlling the disease.1. Three full-length infectious cDNA clones for Japanese, American and English isolates of SBWMV were constructed. Also, an infectivity assay, a barley mesophyl protoplasts system was developed for SBWMV. Reassortment experiments among the three isolates using barley protoplasts showed that all RNA1 and RNA2 combinations supported viral RNA replication, indicating a close relationship among the three geographical isolates.2. Infection of SBWMV to wheat plants requires temperatures lower than 20 ℃. Barley protoplasts inoculated with infectious in vitro transcripts were incubated at temperatures between 15 ℃ and 25 ℃, and it was proved that the optimal temperature for RNA replication is 17 ℃.3. Wild-type RNA2 of SBWMV undergoes frequent deletion mutation when the virus was passaged at 17 ℃ and deletion mutants cause more severe symptoms on wheat plants that the wild-type virus. Deletion mutation was analyzed using full-length infectious RNA2 transcripts in inoculum and found to occur in-frame to the upstream capsid protein gene. Symptom severities correlated with the length of deletion.As described above, the fundamental information, materials and methods were obtained for further detailed analysis on replication, pathogenicity and transmission of furoviruses.