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dc.contributor.authorVan Rijn, Piet A.
dc.contributor.authorVan de Water, Sandra G.P.
dc.contributor.authorFeenstra, Femke
dc.contributor.authorVan Gennip, René G.P.
dc.date.accessioned2017-04-24T09:20:49Z
dc.date.available2017-04-24T09:20:49Z
dc.date.issued2016
dc.identifier.citationVan Rijn, P.A. et al. 2016. Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV). Virology journal, 13(1): Article no 119. [http://dx.doi.org/10.1186/s12985-016-0574-7]en_US
dc.identifier.issn1743-422X (Online)
dc.identifier.urihttp://hdl.handle.net/10394/21540
dc.identifier.urihttp://dx.doi.org/ 10.1186/s12985-016-0574-7
dc.identifier.urihttps://virologyj.biomedcentral.com/articles/10.1186/s12985-016-0574-7
dc.description.abstractBackground 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. Results 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. Conclusions 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 animalsen_US
dc.language.isoenen_US
dc.publisherBioMed Centralen_US
dc.subjectReverse geneticsen_US
dc.subjectOrbivirusen_US
dc.subjectReoviridaeen_US
dc.subjectBluetongue virusen_US
dc.subjectAfrican horse sickness virusen_US
dc.subjectdsRNAen_US
dc.subjectGenetic modificationen_US
dc.subjectReassortmenten_US
dc.titleRequirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV)en_US
dc.typeArticleen_US
dc.contributor.researchID24551287 - Van Rijn, Petrus Antonius


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