Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virus
has a positive sense RNA genome of 11700 bases which encodes for both structural and non structural
proteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria.
Laboratory confirmation is essential as SINV infections must be distinguished from various conditions that
share similar clinical manifestations. The most frequently used methods for identification are
haemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization tests
as well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are not
readily available commercially and due to the non-specific symptoms caused by SINV infection the number
of infections per annum may be under diagnosed. The purpose of this study was to develop serological
assays such as ELISA and a novel neutralization assay that could be used in serological surveys for the
detection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determine
the level of viral replication in mammalian cells for characterizing infection in mammalian cells as well as
investigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINV
infection.
An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitro
neutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISA
and the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-house
ELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity of
the in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical density
values to differentiate positive and negative samples. A forward and reverse primer for the amplification of
a conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with a
TaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammalian
cells, human macrophages and HeLa cells, was determined by measuring viral loads with a real time
quantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strain
from Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viral
replication but were considered insufficient to warrant further investigation and insufficient to investigate
occurrence of antibody dependent enhancement of disease in macrophages. The mechanism possibly
interfering with replication of virus in the human macrophages was investigated.
Supernatant fluid samples from macrophage infections were tested for the release of interferon gamma
which could inhibit viral replication. There were nine to fifteen fold differences in the concentration of interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells were
treated with similar concentrations of human interferon gamma at different time intervals. Pretreatment
and concurrent treatment with infection showed reduced levels of viral load compared with no treatment
or delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viral
replication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNA
fragments separated through agarose gel electrophoreses. There were multiple bands visible in the infected
samples whereas the negative control did not show multiple bands, only one large band of genomic DNA.
The presence of multiple DNA fragments in infected cells and absence of those fragments from uninfected
cells were suggestive of virus induced apoptosis.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-04112014-154931 |
| Date | 11 April 2014 |
| Creators | Hanekom, Hermanus Albertus |
| Contributors | Prof FJ Burt |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-04112014-154931/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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