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North-West University

1. Van der Westhuizen, Maria Jacoba. The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen.

Degree: 2012, North-West University

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa.

Subjects/Keywords: Rotavirus; gastroenteritis; sub-Saharan Africa; G9P[6]; Rotavirus-like particles; transmission electron microscopy; double-layered particles; triple-layered particles,; gastro-enteritis; sub-Sahara Afrika; Rotavirus virusagtige partikels; nie-lewendige entstof; Nonlive vaccine; Baculovirus Expression System; Spodoptera frugiperda; Baculovirus Uitdrukkings Stelsel; transmissie elektron mikroskopie; dubbellaag partikels; trippellaag partikels

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APA (6th Edition):

Van der Westhuizen, M. J. (2012). The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen. (Thesis). North-West University. Retrieved from http://hdl.handle.net/10394/9852

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Van der Westhuizen, Maria Jacoba. “The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen. ” 2012. Thesis, North-West University. Accessed October 24, 2019. http://hdl.handle.net/10394/9852.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Van der Westhuizen, Maria Jacoba. “The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen. ” 2012. Web. 24 Oct 2019.

Vancouver:

Van der Westhuizen MJ. The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen. [Internet] [Thesis]. North-West University; 2012. [cited 2019 Oct 24]. Available from: http://hdl.handle.net/10394/9852.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Van der Westhuizen MJ. The engineering and optimization of expression of rotavirus-like particles in insect cells using a South African G9P[6] rotavirus strain / by Maria J. van der Westhuizen. [Thesis]. North-West University; 2012. Available from: http://hdl.handle.net/10394/9852

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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