Yang Zhang-Nv, Xu Hai-Jun, Thiem Suzanne M, Xu Yi-Peng, Ge Jun-Qing, Tang Xu-Dong, Tian Cai-Hong, Zhang Chuan-Xi
Institute of Insect Science, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, PR China.
J Gen Virol. 2009 Jan;90(Pt 1):162-9. doi: 10.1099/vir.0.004903-0.
The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD(50) was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.
家蚕核型多角体病毒（BmNPV）的ORF9（Bm9）在所有已完成全序列测定的鳞翅目核型多角体病毒中都保守。RT-PCR分析表明，Bm9是一个在感染后3小时开始转录的早期和晚期基因，免疫荧光显微镜检查显示Bm9主要定位于受感染细胞的细胞质中。为了确定Bm9在病毒感染过程中的作用，通过在大肠杆菌中作为杆粒传播的BmNPV基因组中的重组敲除了Bm9。与野生型（wt）杆粒相比，缺失Bm9的杆粒产生的出芽病毒（BV）减少了10倍以上；然而，病毒DNA复制的动力学不受影响。BV产生的缺陷通过Bm9拯救杆粒得以恢复。此外，电子显微镜观察显示多角体的形成不受Bm9缺失的影响。生物测定表明，与wt杆粒相比，缺失Bm9的杆粒杀死五龄家蚕幼虫的时间大约长14 - 22小时，且半数致死剂量（LD50）比wt杆粒高约15倍。总之，Bm9是病毒在体内和体外产生及感染性的一个重要但非必需的因素。
