van Rijn Piet A, van de Water Sandra G P, Feenstra Femke, van Gennip René G P
Department of Virology, Central Veterinary Institute of Wageningen UR (CVI), P.O. Box 65, Lelystad, 8200 AB, The Netherlands.
Department of Biochemistry, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa.
Virol J. 2016 Jul 2;13:119. doi: 10.1186/s12985-016-0574-7.
Bluetongue virus (BTV) and African horse sickness virus (AHSV) are distinct arthropod borne virus species in the genus Orbivirus (Reoviridae family), causing the notifiable diseases Bluetongue and African horse sickness of ruminants and equids, respectively. Reverse genetics systems for these orbiviruses with their ten-segmented genome of double stranded RNA have been developed. Initially, two subsequent transfections of in vitro synthesized capped run-off RNA transcripts resulted in the recovery of BTV. Reverse genetics has been improved by transfection of expression plasmids followed by transfection of ten RNA transcripts. Recovery of AHSV was further improved by use of expression plasmids containing optimized open reading frames.
Plasmids containing full length cDNA of the 10 genome segments for T7 promoter-driven production of full length run-off RNA transcripts and expression plasmids with optimized open reading frames (ORFs) were used. BTV and AHSV were rescued using reverse genetics. The requirement of each expression plasmid and capping of RNA transcripts for reverse genetics were studied and compared for BTV and AHSV. BTV was recovered by transfection of VP1 and NS2 expression plasmids followed by transfection of a set of ten capped RNAs. VP3 expression plasmid was also required if uncapped RNAs were transfected. Recovery of AHSV required transfection of VP1, VP3 and NS2 expression plasmids followed by transfection of capped RNA transcripts. Plasmid-driven expression of VP4, 6 and 7 was also needed when uncapped RNA transcripts were used. Irrespective of capping of RNA transcripts, NS1 expression plasmid was not needed for recovery, although NS1 protein is essential for virus propagation. Improvement of reverse genetics for AHSV was clearly demonstrated by rescue of several mutants and reassortants that were not rescued with previous methods.
A limited number of expression plasmids is required for rescue of BTV or AHSV using reverse genetics, making the system much more versatile and generally applicable. Optimization of reverse genetics enlarge the possibilities to rescue virus mutants and reassortants, and will greatly benefit the control of these important diseases of livestock and companion animals.
蓝舌病毒（BTV）和非洲马瘟病毒（AHSV）是呼肠孤病毒科环状病毒属中不同的节肢动物传播病毒种，分别引起反刍动物的蓝舌病和马属动物的非洲马瘟这两种应通报疾病。针对这些具有双链RNA十片段基因组的环状病毒，已开发出反向遗传系统。最初，对体外合成的加帽连续RNA转录本进行两次连续转染可获得BTV。通过转染表达质粒，随后转染十个RNA转录本，反向遗传学得到了改进。使用含有优化开放阅读框的表达质粒，进一步提高了AHSV的拯救效率。
使用了含有10个基因组片段全长cDNA的质粒，用于T7启动子驱动全长连续RNA转录本的产生，以及含有优化开放阅读框（ORF）的表达质粒。利用反向遗传学拯救了BTV和AHSV。研究并比较了BTV和AHSV反向遗传学中每种表达质粒的需求以及RNA转录本的加帽情况。通过转染VP1和NS2表达质粒，随后转染一组十个加帽RNA来拯救BTV。如果转染未加帽的RNA，则还需要VP3表达质粒。拯救AHSV需要转染VP1、VP3和NS2表达质粒，随后转染加帽RNA转录本。当使用未加帽的RNA转录本时，还需要质粒驱动的VP4、6和7的表达。无论RNA转录本是否加帽，拯救过程都不需要NS1表达质粒，尽管NS1蛋白对病毒传播至关重要。通过拯救几种用以前方法无法拯救的突变体和重配体，清楚地证明了AHSV反向遗传学的改进。
使用反向遗传学拯救BTV或AHSV所需的表达质粒数量有限，使该系统更加通用且普遍适用。反向遗传学的优化扩大了拯救病毒突变体和重配体的可能性，并将极大地有利于控制这些影响家畜和伴侣动物的重要疾病。
