Preparation of a set of rotavirus SA 11 transcription plasmids for T7- transcript based reverse genetics

Abstract

Rotaviruses belong to the Reoviridae family, and rotavirus infection is the biggest contributor to diarrhoea-related death in the world for children under the age of five years. The rotavirus genome consists of 11 double-stranded RNA segments and is build into a triple-layer particle. The 11 genome segments encode six structural proteins VP1, VP2, VP3, VP4, VP6 and VP7 together with six non-structural proteins NSP1, NSP2, NSP3, NSP4 and NSP5/6. Reverse genetics are biological methods that are used to generate insights into the workings and characteristics of pathogenesis and the replication cycle of viruses. Reverse genetics systems have been established for several dsRNA viruses such as Bluetongue virus (BTV) and African horsesickness virus (AHSV) and is used to develop better vaccines for these viruses. It was not until early 2017 when a plasmid-based reverse genetics system for rotavirus was developed (Kanai et al., 2017), and there is currently still no rotavirus transcript-based reverse genetics system. This project aimed to develop such a transcript-based reverse genetics system for rotavirus by incorporating different aspects of reverse genetics systems of BTV, AHSV and the plasmid-based system of rotavirus. To achieve this a design flaw in four rotavirus multiple genome segment plasmids from a previous study had to be corrected. This design flaw had three additional guanines at the 5' terminal ends of all 11 genome segments which led to the (+)ssRNAs to not be packaged. The design was corrected through In-Fusion HD cloning which is a state-of-the-art cloning method that allows cloning of one or multiple DNA fragments into any vector of choice at any position, provided there is a 15-base pair overlap on both ends of the vector and DNA fragment. The 15-base overlap was generated with PCR with specifically designed primers, and the 5' and 3' terminal ends were joined with the In-Fusion enzyme creating 10 rotavirus transcription plasmids pSMART-GS1/2/4/5/6/7/8/9/10/11. After multiple failed attempts to clone genome segment 3 into pSMART, it was decided to correct the design flaw for this genome segment with PCR and use the amplicon to synthesise (+)ssRNAs. To determine if the initial design flaw of three extra guanine nucleotides were successfully removed and that the respective 5' and 3' ends annealed correctly, the transcription plasmids were sent for Sanger sequencing. In addition, the transcription plasmids underwent next-generation sequencing to determine if any nucleotide changes had occurred in the sequences of the transcription plasmids. The results of the Ion-Torrent S5 sequencing showed a nucleotide change from a thymine to a cytosine in genome segment 11 at position 289. This change in sequence would invoke a change in amino acid from a cysteine to an arginine (C289R). However, due to time limitations, we had to proceed with this error. The transcription plasmids were used to synthesise (+)ssRNAs through in vitro transcription. The identity of the (+)ssRNAs was confirmed with agarose gel electrophoresis. Finally, the 11 newly synthesised (+)ssRNAs together with the fusogenic orthoreovirus FAST plasmid, two vaccinia virus capping enzyme plasmids (D1R and D12L) and seven rotavirus expression plasmids encoding the replication complex and viroplasm (VP1, VP2, VP3, VP6, NSP1, NSP2 and NSP5) was transfected into BHK-T7 cells with Lipofectamine® 2000. After 22 hours the BHK-T7 cells were co-seeded with MA104 cells, and after 7 days of incubation, no cytopathic effect (CPE) was observed. An immunofluorescence monolayer assay (IMFA) was conducted on the co-seeded cell monolayer to determine if any rotavirus was rescued. However, no fluorescence was observed. The lack of rescue was attributed to the nucleotide change in genome segment 11 and the overuse of the FAST plasmid during transfection. Thus, this attempt to establish a rotavirus transcript-based reverse genetics system was unsuccessful, but the transcription plasmids should be useful for future experiments.