Rescue of Rabies Virus from Cloned cDNA and Identification of the Pathogenicity-Related Gene: Glycoprotein Gene Is Associated with Virulence for Adult Mice

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.

为鉴定与狂犬病病毒致病性相关的病毒基因，本研究尝试构建减毒RC-HL株的反向遗传操作系统。该毒株经脑内接种后，可在成年小鼠体内引发非致死性感染。研究人员构建了编码RC-HL株完整抗原组cDNA的全长基因组质粒，以及分别包含N、P、L基因完整开放阅读框对应cDNA的辅助质粒。将上述质粒转染至被表达T7 RNA聚合酶（T7 RNA polymerase）的痘苗病毒（vaccinia virus）感染的BHK-21细胞后，成功拯救得到与野生型RC-HL株生物学特性近乎一致的传染性狂犬病病毒。利用该RC-HL株反向遗传操作系统，本研究在RC-HL基因组背景下，制备了一株嵌合病毒，其糖蛋白基因开放阅读框来自亲本Nishigahara株——该亲本株经脑内接种后可致死成年小鼠。由于该嵌合病毒经脑内接种后可导致成年小鼠死亡，由此证实，糖蛋白基因的开放阅读框与Nishigahara株对成年小鼠的致病性密切相关。