Interaction of alphaviruses chikungunya and Semliki Forest with cells of the mononuclear phagocyte system
Zagrajek, Adrian Krzysztof
Introduction Chikungunya virus (CHIKV) is an alphavirus in the family Togaviridae. Since 2005 the virus has caused a major epidemic of disease in humans, ranging from Central Africa, South-East Asia, Caribbean and more recently the Americas. The virus is spread by mosquitoes, most notably Aedes aegypti and Ae. albopictus. CHIKV causes an acute disease in humans, which is characterised by a rapid onset of high fever, rash, myalgia and arthralgia. The symptoms typically resolve within a week. Remarkably, up to a third of patients who recover from acute chikungunya develop chronic arthritis/arthralgia, which may last for months or years and has a large negative impact on the quality of life. The mechanism by which this occurs is not yet fully understood. CHIKV can infect human monocytes, and macrophages positive for CHIKV antigen have been observed in joint tissue from patients recovered from acute CHIKV infection but with chronic arthritis. Furthermore, it has been demonstrated that macrophages can be infected with CHIKV in vitro by a mechanism involving apoptotic debris from CHIKV-infected cells. Hypothesis and aims Infection of monocytes and macrophages with CHIKV contributes to clinical disease and virus persistence in vivo. The aim of this project was to investigate the mechanism by which alphaviruses infect macrophages in vitro, and to generate a CHIKV which is unable to replicate in monocytes and macrophages in vitro, and to study its pathogenicity in vivo. Materials and methods HeLa cells were infected with Semliki Forest virus (SFV), an alphavirus closely related to CHIKV, or SFV replicon particles (SFV VRP). Following cell death, whole cell supernatant or clarified cell supernatant from SFV- and SFV VRP-infected cells was passaged onto human monocyte-derived macrophages (MDMs). These cells were observed microscopically for expression of the fluorescent marker encoded by the SFV. Virus and VRP-infected apoptotic debris were inspected for the presence of alphavirus replication complexes by electron microscopy. Subsequently, a recognition element (RE) for a haematopoietic-specific miRNA (miR-142-3P) was incorporated into the genome of SFV (proof-of-concept) and CHIKV to investigate if blocking virus replication in cells of the mononuclear phagocyte system altered virus kinetics in vitro. The replication of the modified viruses was investigated in macrophage/monocyte cell lines Thp-1 and IC-21, and in HEK 293 cells modified to express miR-142-3P under the control of an inducible tetracycline promoter. Modified viruses were tested in animal models of disease (mouse for SFV and non-human primate for CHIKV) to investigate the pathogenicity of these viruses in vivo. Results The presence of apoptotic debris from SFV-infected cells was required to infect MDMs with SFV. The presence or absence of infectious virus particles in the apoptotic debris did not affect the infection rate. Intact alphavirus replication complexes were found within the apoptotic debris. MiR-142-3P RE was successfully incorporated into the genome of both SFV and CHIKV. RE-virus replication in all cells expressing miR-142-3P was reduced by 90-99% when compared to control viruses. RE-virus replication was not affected in cells which did not express miR- 142-3P. In interferon-α/β receptor knockout mice, RE-SFV generated viraemia comparable to the control virus, but could not infect efficiently the population of macrophages resident in the marginal zone of the spleen. RE-CHIKV was found to be genetically stable in vitro following multiple passages on BHK-21 cells in the absence of a selective pressure from miR-142-3P. RE-CHIKV was inoculated into two cynomolgus macaques. The data from this experiment are not yet available. Conclusion SFV was shown to infect MDM via apoptotic debris containing intact alphavirus replication complexes, which were the most likely infectious agent. SFV and CHIKV unable to replicate in haematopoietic cells in vitro were successfully engineered. The pathogenicity of modified SFV and CHIKV was investigated in vivo.