鸭坦布苏病毒非编码亚基因组RNA的鉴定及其影响病毒致病性的分子机制研究

Duck Tembusu virus (DTUMV) is a member of the genus Flavivirus and the family Flaviviridae, which can cause severely decreased egg production and/or severe neurological disorders bringing huge economic losses in the Chinese duck industry. The recent studies indicated the flavivirus produce a unique and highly structured noncoding subgenomic RNA (sgRNA) of 0.3–0.5 kb derived from the genome during viral replication, which is essential for virus-induced cytopathicity and pathogenicity. However, until now, there remains no report of identification and functional studies on DTUMV sgRNA. The present study employed the high throughput deep sequencing, northern blot hybridization, exoribonuclease XRN1 treatment to identify sgRNAs sequences. By aligning the sgRNA sequences to the TMUV 3’ UTR, the exactly sgRNAs targeted regions were finally obtained. Furthermore, Our previously constructed DTUMV full-length cDNA infectious clone were used for engineering the recombinant DTUMV with the deletion of the corresponding partial 3’ UTR region which cannot produce the sgRNAs during the viral replication. In vitro, the viral pathogenesis and relative viral gene expression were tested to reveal the sgRNAs has a role in facilitating efficient viral genome replication and virus-induced cytopathicity in cell culture. In vivo, the pathogenicity of the sgRNA deleted recombinant DTUMV were tested by inoculating the ducklings and layer ducks and compared with the sgRNA undeleted DTUMV to reveal the sgRNA has a role in the DTMUV pathogenesis and host antiviral mechanism. This study will not only clarify the sgRNA is essential for the viral replication, virus-induced cytopathicity, pathogenicity, escape host antiviral mechanism; but also provide a basis for the further development of an sgRNA deleted recombinant DTUMV based attenuated vaccine.

鸭坦布苏病毒(DTMUV)可导致鸭严重的产蛋下降和神经症状，严重危害我国养鸭业的健康发展。研究表明，黄病毒在复制过程中, 基因组的3’ 非编码区会产生约0.3-0.5kb具有调节病毒致病性作用的非编码亚基因组RNA（sgRNA）。目前有关DTMUV的sgRNA的鉴定和功能研究国内外均未见报道。本研究采用高通量测序、分子杂交以及核糖核酸酶XRN1处理技术对DTMUV的sgRNA进行鉴定，明确sgRNA在病毒基因组中的准确定位；对构建的DTMUV感染性克隆的sgRNA锚定区域进行突变，拯救出无sgRNA产生能力的DTMUV突变株；通过突变株体外试验分析sgRNA在致细胞病变及其对病毒基因组复制的调控作用；通过突变株产蛋鸭感染实验阐明sgRNA与致病性及宿主抗病毒机制的相关性。本项目为DTUMV的sgRNA缺失候选疫苗株的研究提供了理论依据。

鸭坦布苏病毒(DTMUV)可导致鸭严重的产蛋下降和神经症状，严重危害我国养鸭业的健康发展。研究表明，黄病毒在复制过程中，基因组的3’非编码区会产生约0.3-0.5kb具有调节病毒致病性作用的非编码亚基因组RNA（sgRNA）。本项目主要以sgRNA的鉴定及影响病毒致病性的分子机制为基础，重点探讨sgRNA在病毒复制、细胞毒性、致病性及病毒逃逸宿主抗病毒机制中的作用。本研究采用高通量测序、分子杂交以及核糖核酸酶XRN1处理技术DTMUV的sgRNA进行鉴定，准确定位出TMUV产生的sgRNA位于病毒基因组10463-10990处，长度约为528nt；对构建的DTMUV感染性克隆的sgRNA锚定区域进行突变，构建了4株sgRNA突变株，经sgRNA鉴定分析后，成功拯救出无sgRNA产生能力的DTMUV突变株2株；突变株转染细胞后，与原始毒株毒力、生长曲线进行对比，确定了突变株可降低病毒的复制能力；此外，体外过表达sgRNA后接种DTMUV24h和36h对NS1基因表达量的分析显示，过表达sgRNA后，病毒NS1基因表达量显著升高，确定了sgRNA为DTMUV介导细胞病变产生的必要元件。以上研究阐明了sgRNA在DTMUV复制、致病过程中的重要作用，为研发DTMUV sgRNA缺失候选疫苗株的研究提供了理论依据。