In vitro immune responses to Sindbis virus
| dc.contributor.advisor | Burt, Felicity | |
| dc.contributor.author | Litabe, Matefo Millicent | |
| dc.date.accessioned | 2020-12-07T07:28:11Z | |
| dc.date.available | 2020-12-07T07:28:11Z | |
| dc.date.issued | 2020-02 | |
| dc.description.abstract | Sindbis virus (SINV) is an arthritogenic alphavirus belonging to the Togaviridae family. SINV was initially isolated in Sindbis, Egypt in 1952, since then the virus has been isolated in different parts of the world including Africa, Asia, Australia and Europe. Infection with SINV results in febrile self-limiting symptoms that are usually of short duration. However, some individuals develop prolonged incapacitating joint pain that may persist for months to years. The mechanism by which SINV and other arthritogenic alphaviruses cause chronic arthritis is poorly understood however previous studies suggest the involvement of monocytes and macrophages which result in the secretion of pro-inflammatory cytokines which are induced by virus replicating in or around joint tissue. The aim of the study was to investigate the innate immune response to in vitro infection of macrophages with SINV in order to determine if human macrophages from different individuals differ in their susceptibility to SINV infection and to determine the role of interferon (IFN) in SINV infected macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from ten SINV antibody naïve individuals using ficoll-paque density gradient method. The cells were stimulated with human-macrophage colony-stimulating factor (HM-CSF) to differentiate from monocytes to macrophages. Post-stimulation the macrophages were infected with SINV at a multiplicity of infection (MOI) of 0.1. Additionally, some macrophages cultures were pre-treated with ruxolitinib, an IFN inhibitor 2 hours or immediately prior to infection with SINV at an MOI of 0.1. Virus replication was determined at different time intervals for 24 hours using a two-step quantitative RT-PCR. To determine the secretion levels of pro-inflammatory cytokines post-SINV infection, cell-free supernatant was collected at different intervals post-infection and levels of proinflammatory cytokines including IFN-α, TNF-α, IL-1β, IL-6, IL-8 and IL-12 in the supernatant were tested by ELISA. A primer pair that targets the nsP2 region of SINV nsP2 protein together with the TaqMan hydrolysis probe were used to quantify viral loads of infected macrophages by qRT-PCR. An increase in viral loads was detected in macrophages of 5/10 participants, suggestive of viral replication. A decrease in viral load over time was observed in macrophages of the remaining five participants, suggestive of little to no viral replication. IFN inhibition resulted in SINV replication in macrophages from 7/10 and 8/10 participants when treated at time 0 of infection and 2 hours prior to infection, respectively. Infection elicited a strong innate immune response demonstrated by secretion of pro-inflammatory cytokines including IFN-α. IL-6 and IL-8. Infection with SINV results in a strong pro-inflammatory response which seems to control viral load. Results suggest that macrophages are among SINV targeted cells during human infection and macrophages from different individuals appear to differ in their susceptibility to SINV replication. The study also shows that type I IFN may play an important role in the protection of SINV as its inhibition rendered macrophages from more participants susceptible to viral replication. | en_ZA |
| dc.description.sponsorship | National Health Laboratory Service | en_ZA |
| dc.description.sponsorship | The South African Chairs initiative National Research Foundation | en_ZA |
| dc.description.sponsorship | Poliomyelitis Research Foundation | en_ZA |
| dc.description.sponsorship | University of the Free State School of Medicine | en_ZA |
| dc.description.sponsorship | University of the Free State Postgraduate School | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/10859 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Dissertation (M.Med.Sc. (Medical Microbiology and Virology))--University of the Free State, 2020 | en_ZA |
| dc.subject | Immune response - Sindbis virus. | en_ZA |
| dc.subject | Immune response to parasitic infections | en_ZA |
| dc.subject | In vitro infection of macrophages with SINV | en_ZA |
| dc.title | In vitro immune responses to Sindbis virus | en_ZA |
| dc.type | Dissertation | en_ZA |