Reverse genetics is an innovative molecular biology tool that enables the manipulation of
viral genomes at the cDNA level in order to generate particular mutants or artificial viruses.
The reverse genetics system for the influenza virus is arguably one of the best illustrations of
the potential power of this technology. This reverse genetics system is the basis for the
ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have
been developed for many animal RNA viruses. Selection-free reverse genetics systems have
been developed for the members of the Reoviridae family including, African horsesickness
virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the
generation of valuable evidence regarding the replication and pathogenesis of these viruses.
Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics
systems to rotavirus has not yet been successful. The development of a selection-free
rotavirus reverse genetics system will enable the systematic investigation of poorly
understood aspects of the rotavirus replication cycle and aid the development of more
effective vaccines, amongst other research avenues.
This study investigated the importance of co-expressed rotavirus proteins in the
development of a selection-free rotavirus reverse genetics system. The consensus
sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11
(RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression
plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was
determined by sequence-independent cDNA synthesis and amplification combined with
next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also
resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome
segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of
the detected nucleotide changes, and consequent amino acid variations, had any significant
effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS
was closely related to the ParWa and VirWa variants, which were derived from the original
1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome
seems to be stable. Considering that the current reference sequence for the Wa strain is a
composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate
reference sequence.
The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus
proteins, under control of a T7 promoter sequence, due to the fact that they propagate well
in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase
was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a
variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression
rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2
and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another
approach involved the codon-optimised expression of the rotavirus replication complex
scaffold in MA104 cells under the control of a CMV promoter sequence. This system was
independent from the recombinant fowlpox virus. All three plasmid expression sets were
designed to be used in combination with the transcript-based reverse genetics system in
order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double
layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the
expression of rotavirus transcripts although expression of rotavirus VP6 could be
demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using
immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and
NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is
the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell
death pattern was observed within 24 hours in response to transfection of rotavirus
transcripts. This observed cell death, however does not seem to be related to normal viral
cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the
transcript- and plasmid systems, a dual transfection strategy was followed where plasmids
encoding rotavirus proteins were transfected first followed, 12 hours later, by the
transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was
designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6),
the rotavirus replication complex would form and assist with replication and/or packaging.
Transfecting codon- optimized plasmids first noticeably delayed the mass cell death
observed when transfecting rotavirus transcripts on their own. None of the examined coexpression
systems were able to produce a viable rotavirus.
Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived
rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR)
experiments indicated that rotavirus transcripts induced high levels of the expression of the
cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from
plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses,
while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the
expression of certain cytokines. In the light of these suppression results, specific rotavirus
proteins were expressed from transfected plasmids to investigate their potential in
supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results
indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of
NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by
rotavirus transcripts. These findings point to other possible viral innate suppression
mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The
suppression of the strong innate immune response elicited by rotavirus transcripts might
well prove to be vital in the quest to better understand the replication cycle of this virus and
eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/12270 |
| Date | January 2014 |
| Creators | Wentzel, Johannes Frederik |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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