Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Virus Res. 2011 Sep;160(1-2):166-72. doi: 10.1016/j.virusres.2011.06.007. Epub 2011 Jun 16.
Chandipura virus (CV; genus Vesiculovirus, family Rhabdoviridae) is an emerging arbovirus, responsible for a number of outbreaks of severe viral encephalitis affecting children in India. A reverse genetics system has been constructed which allows recovery of infectious recombinant CV (rCV) entirely from cDNA. This system was used to construct a virus, rCVE, which has an additional transcription unit encoding green fluorescent protein (EGFP) between the 3rd and 4th CV genes. This virus grew to titres comparable to the parental rCV and stably expressed EGFP in infected cells for at least 4 passages. A second virus, rCVGM, was constructed in which the CV matrix (M) coding region was replaced with the coding region for an EGFP-M fusion protein. Compared to rCV and rCVE, rCVGM was attenuated, giving a small plaque phenotype and lower yield, although it did express the EGFP-M protein in infected cells. Passage of rCVGM resulted in viruses with a standard plaque phenotype which no longer expressed EGFP. Analysis of two of these viruses showed that most or all of the EGFP ORF was deleted. The EGFP-M fusion protein showed cleavage to EGFP-sized and M-sized products, both in rCVGM-infected cells and when expressed from a plasmid. The EGFP-M fusion protein was not detected in virus particles, suggesting it was incompatible with virus assembly, and particles of rCVGM likely contained the M-sized cleavage product in its place.
钱德普拉病毒（CV；属 Vesiculovirus，Rhabdoviridae 科）是一种新兴的虫媒病毒，可引起印度儿童发生多起严重病毒性脑炎暴发。现已构建出一种可完全从 cDNA 恢复感染性重组 CV（rCV）的反向遗传学系统。该系统用于构建一种病毒 rCVE，该病毒在第 3 和第 4 个 CV 基因之间插入了一个编码绿色荧光蛋白（EGFP）的额外转录单元。该病毒的滴度与亲本 rCV 相当，在感染细胞中至少可稳定表达 4 个传代的 EGFP。第二个病毒 rCVGM 构建时，CV 基质（M）编码区被 EGFP-M 融合蛋白编码区取代。与 rCV 和 rCVE 相比，rCVGM 减毒，形成小斑表型，产量降低，尽管它在感染细胞中确实表达了 EGFP-M 蛋白。rCVGM 的传代导致出现具有标准斑表型且不再表达 EGFP 的病毒。对其中两种病毒的分析表明，EGFP ORF 大部分或全部缺失。EGFP-M 融合蛋白在 rCVGM 感染的细胞中以及从质粒表达时均被切割成 EGFP 大小和 M 大小的产物。在病毒颗粒中未检测到 EGFP-M 融合蛋白，表明其与病毒装配不兼容，rCVGM 的颗粒可能以 M 大小的切割产物代替。
