Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, People's Republic of China.
Virus Res. 2011 Sep;160(1-2):439-43. doi: 10.1016/j.virusres.2011.07.008. Epub 2011 Jul 27.
Recently, duck hepatitis A virus 3 (DHAV-3) with genetically distinct characteristics from DHAV-1 and DHAV-2 was recognized in South Korea and China. In this short communication, we successfully constructed a stable full-length infectious cDNA clone derived from DHAV-3 by solving instability of cloned full-length cDNA in Escherichia coli (E. coli). The cDNA fragments amplified from the genome of DHAV-3 were assembled and inserted into a low-copy-number plasmid. Finally, a full-length cDNA clone containing an engineered SacII site that served as a genetic marker was obtained. The cDNA clone showed stable by serial passages in E. coli when propagated at 25°C under low level of antibiotic selection. BHK-21 cells were transfected with transcribed RNA from the full-length cDNA clone; infectious viral particles were rescued, showing its fatality to 10-day-old duck embryos. The results indicated that the constructed full-length cDNA clone of DHAV-3 is infectious. By various virological assays, our results indicated that the rescued virus exhibited similar biological properties with the parental virus. Animal experiments revealed that the rescued virus retained the high pathogenicity to 1-day-old ducklings and could induce a fatal hepatitis indistinguishable from its parental virus. Our present studies provide a useful tool for future research on genomic functions and molecular pathogenesis of DHAV-3.
最近，在韩国和中国发现了一种与 DHAV-1 和 DHAV-2 具有明显遗传特征的鸭肝炎 A 病毒 3（DHAV-3）。在本简讯中，我们通过解决大肠杆菌（E. coli）中克隆全长 cDNA 的不稳定性，成功构建了源自 DHAV-3 的稳定全长感染性 cDNA 克隆。从 DHAV-3 基因组扩增的 cDNA 片段被组装并插入低拷贝数质粒中。最终，获得了包含工程化 SacII 位点的全长 cDNA 克隆，该位点作为遗传标记。当在 25°C 下以低水平抗生素选择进行繁殖时，cDNA 克隆在大肠杆菌中连续传代时表现稳定。BHK-21 细胞用全长 cDNA 克隆转录的 RNA 转染；拯救出感染性病毒颗粒，表明其对 10 日龄鸭胚具有致死性。结果表明，构建的 DHAV-3 全长 cDNA 克隆是感染性的。通过各种病毒学检测，我们的结果表明，拯救的病毒表现出与亲本病毒相似的生物学特性。动物实验表明，拯救的病毒保持了对 1 日龄雏鸭的高致病性，并能引起与亲本病毒无法区分的致命肝炎。我们目前的研究为未来研究 DHAV-3 的基因组功能和分子发病机制提供了有用的工具。
