Guo Rui, Cao Guangli, Zhu Yuexiong, Kumar Dhiraj, Xue Renyu, Lu Yahong, Hu Xiaolong, Gong Chengliang
College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China.
Curr Microbiol. 2016 Oct;73(4):587-94. doi: 10.1007/s00284-016-1102-0. Epub 2016 Jul 22.
Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.
家蚕双顺反子病毒（BmBDV）先前被称为家蚕浓核病毒2型，后来它被重新分类到新科双顺反子病毒科的新属双顺反子病毒中。家蚕双顺反子病毒镇江分离株（BmBDV-Z）的基因组由两个非同源的单链线性DNA分子VD1和VD2组成，它们被包装到单独的病毒粒子中。为了研究BmBDV DNA的感染性，将插入VD1基因组的重组质粒pGEM-VD1转染到家蚕的BmN细胞中。通过蛋白质免疫印迹法和免疫荧光测定法检测BmBDV的结构蛋白，这表明pGEM-VD1在转染的BmN细胞中复制并且病毒蛋白也得以表达。通过透射电子显微镜观察，我们鉴定出了约20纳米的类BmBDV病毒粒子，这证实了转染后可产生BmBDV。随后，构建了插入VD1基因组的重组杆状病毒BmBac-VD1。蛋白质免疫印迹法和免疫荧光测定法的结果表明，BmBDV的病毒结构蛋白在BmBac-VD1感染的细胞中表达。通过透射电子显微镜在感染的细胞中还观察到了杆状和球形病毒粒子，并且两种病毒粒子被分离出来。然而，分子生物学检测结果显示，来自BmBac-VD1的感染性序列被包装在球形病毒粒子内。因此，我们认为插入BmBDV基因组DNA的载体具有感染性，并且在培养的BmN细胞中可以产生包装重组DNA的类BmBDV病毒粒子。我们当前研究的结果不仅提供了一种在体外探索BmBDV基因功能的新感染方法，而且还提供了一个有助于开发更有效新型杀虫剂的方案。
