Reverse genetics systems that are based on either viral transcripts or cDNA genome
segments cloned in plasmids have recently been reported for some of the dsRNA
viruses of the Reoviridae family, namely African horsesickness virus, bluetongue
virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only
allow the manipulation of a single genome segment have been described. These
rotavirus single genome segment reverse genetics systems are not true stand-alone
systems because they require a helper virus and a recombinant virus selection step.
A true selection-free, plasmid- only or transcript-based reverse genetics system for
rotaviruses is lacking.
This study sought to identify and characterise the factors that need to be understood
and overcome for the development of a rotavirus reverse genetics system using
mRNA derived from the in vitro transcription of a consensus nucleotide sequence as
well as from double-layered particles. The consensus whole genome sequence of
the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent
whole genome amplification and 454® pyrosequencing. For the
rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at
position 397 in a hydrophobic region of VP4. NSP1 contained seven additional
amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus
nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'-
terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10
(NSP4) of the DS-1 strain were determined in this study. The consensus genome
segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously
reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known
passage history revealed a mixed infection with two SA11 strains. One of the strains
was a reassortant which contained genome segment 8 (NSP2) from the bovine
rotavirus O agent. The other ten consensus genome segments of the two strains
could not be differentiated. Novel minor population variants of genome segments 4
(VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic
analyses of the rotavirus SA11 genomes showed that the two SA11 strains were
closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were
purchased and used to generate exact capped transcripts by in vitro transcription
with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in
vitro transcription using purified rotavirus SA11 double-layered particles. The purified
rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104
cells. Work on MA104 cells was discontinued due their very low transfection efficacy.
In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death.
However, no viable rotavirus was recovered following attempts to infect MA104 cells
with the BSR and COS-7 transfected cell lysates. The cell death was determined to
be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1
genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR
and COS-7 cells. Based on visual inspection, the translation seemed to be higher in
the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells.
This suggested that the transfection of rotavirus transcripts induced an innate
immune response which could lead to the development of an antiviral state.
Therefore, the innate immune response to rotavirus transcripts was investigated in
HEK 293H cells using qRT-PCR and western blot analyses. Results of this
investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in
transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of
cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other
cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly
induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction
of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not
abrogated. The importance of a consensus sequence and the insights gained in the
current study regarding the role of the innate immune response after transfection of
rotavirus transcripts into cells in culture, should aid the development of a true
rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/9523 |
| Date | January 2012 |
| Creators | Mlera, Luwanika |
| Publisher | North-West University |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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