Development of detection assays for sindbus virus and investigating in vitro infection of mammalian cells

[摘要] Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virushas a positive sense RNA genome of 11700 bases which encodes for both structural and non structuralproteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria.Laboratory confirmation is essential as SINV infections must be distinguished from various conditions thatshare similar clinical manifestations. The most frequently used methods for identification arehaemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization testsas well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are notreadily available commercially and due to the non-specific symptoms caused by SINV infection the numberof infections per annum may be under diagnosed. The purpose of this study was to develop serologicalassays such as ELISA and a novel neutralization assay that could be used in serological surveys for thedetection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determinethe level of viral replication in mammalian cells for characterizing infection in mammalian cells as well asinvestigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINVinfection.An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitroneutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISAand the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-houseELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity ofthe in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical densityvalues to differentiate positive and negative samples. A forward and reverse primer for the amplification ofa conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with aTaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammaliancells, human macrophages and HeLa cells, was determined by measuring viral loads with a real timequantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strainfrom Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viralreplication but were considered insufficient to warrant further investigation and insufficient to investigateoccurrence of antibody dependent enhancement of disease in macrophages. The mechanism possiblyinterfering with replication of virus in the human macrophages was investigated.Supernatant fluid samples from macrophage infections were tested for the release of interferon gammawhich could inhibit viral replication. There were nine to fifteen fold differences in the concentration of 2interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells weretreated with similar concentrations of human interferon gamma at different time intervals. Pretreatmentand concurrent treatment with infection showed reduced levels of viral load compared with no treatmentor delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viralreplication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNAfragments separated through agarose gel electrophoreses. There were multiple bands visible in the infectedsamples 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 uninfectedcells were suggestive of virus induced apoptosis.

[发布机构] University of the Free State