Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and
RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young
mammals and the need for further development of additional rotavirus vaccines, especially
vaccines effective against regional strains in developing country settings, is increasing. The
design and formulation of new effective multivalent rotavirus vaccines is complicated by the
wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the
propensity of rotaviruses to evolve using mechanisms such as point mutation, genome
segment reassortment, genome segment recombination and interspecies transmission.
Mutations occurring within the primer binding regions targeted by the current commonly
employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel
mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with
online rotavirus genotyping tools will help to understand the complete epidemiology of the
circulating strains which, in turn, is vital for developing intervention measures such as
vaccine and anti-viral therapies.
In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides
that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing,
and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole
genome of rotaviruses. The robustness of this approach was demonstrated in characterising
the complete genetic constellations and evolutionary origin of selected human rotavirus
strains that emerged in the past two decades worldwide, human rotavirus strains frequently
detected in Africa, and the whole genomes of some common strains frequently detected in
bovine species. Most of the characterised strains emerged either through intra- or interspecies
genome segment reassortment processes. The methods used in this study also allowed
determination of the whole consensus genome sequence of multiple rotavirus variants present
in a single stool sample and the elucidation of the evolutionary mechanisms that explained
their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype
rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2)
and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent
amplification of the rotavirus genomes allowed the determination of the consensus nucleotide
sequence for each of the genome segments of the selected study strains directly from stool
sample.
The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and
VP7 of some of the study strains were codon optimised for insect cell expression and used to
generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used
to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs
contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/
ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of
various combinations of VP4 and VP7 were assembled. The outer capsid proteins were
derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or
P[8] genotypes that were directly extracted from human stool faecal specimens. The
structures of these chimaeric RV-VLPs were morphologically evaluated using transmission
electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered
(dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant
rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the
assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs.
These RV-VLPs will be evaluated in future animal studies as potential non-live
rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this
study, namely by using the consensus nucleotide sequence derived from dsRNA extracted
directly from clinical specimens, should speed up vaccine research and development by
bypassing the need to adapt the viruses to tissue culture and circumventing some other
problems associated with cell culture adaptation as well. Thus, it is now possible to generate
RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field
rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/8502 |
| Date | January 2012 |
| Creators | Jere, Khuzwayo Chidiwa |
| Publisher | North-West University |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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