Middle East respiratory syndrome : the disease and the virus

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) has infected >2000 patients with >30% fatality since 2012. This thesis aimed to develop novel diagnostics, identify effective treatments, and characterize new pathogenic mechanisms of MERS.

To characterize the replication kinetics and tissue tropism of MERS-CoV, the virus was cultured in cell lines of different tissue origins. Unlike other coronaviruses which are difficult to culture in vitro, MERS-CoV replicated rapidly in many cell lines. These findings correlated with the protean clinical manifestations of MERS.

The abundantly expressed leader sequences at the 5’-untranslated region of MERS-CoV and other coronaviruses were used as novel diagnostic targets in newly developed real-time quantitative reverse transcription-polymerase chain reaction assays. Locked nucleic acid probes were employed to overcome the short lengths of the leader sequences. These assays were equally specific and more sensitive for detecting coronaviruses in patients’ nasopharyngeal aspirates than a commonly used commercial multiplex assay (ResPlex-II Panel v2.0).

New immunofluorescent and neutralizing antibody detection assays for MERS were developed. Unexpectedly, cross-reactivity was observed between SARS patients’ sera and MERS-CoV in both immunofluorescent antibody and virus neutralization tests. Bioinformatic analysis showed that the cross-reactivity was likely related to an epitope around the heptad repeat 2 domain of the viral spike protein. These results had direct impact on the use and interpretation of antibody detection assays for MERS.

A sensitive and specific antigen detection assay for MERS was developed. The assay’s low limit of detection and ease of use should make it useful for high-throughput testing of human and animal samples in epidemic regions.

Specific anti-MERS treatments would take years to develop. Repurposing clinically approved drugs with anti-MERS-CoV activity provided immediately available treatment options. A drug repurposing program was conducted to identify the in vitro anti-MERS-CoV effects of recombinant interferons, mycophenolic acid, and ribavirin. A nonhuman primate common marmoset model for MERS was then used to validate the in vivo treatment effects of lopinavir/ritonavir and interferon-β1b. These novel findings provided the basis for clinical trials using lopinavir/ritonavir, recombinant interferons, and/or ribavirin to treat MERS patients in the Middle East and elsewhere.

Using a combination of in vitro, ex vivo, and in vivo models, unique pathogenic mechanisms utilized by MERS-CoV to overcome the host innate and adaptive immune responses and disseminate systemically to cause organ damage were characterized. First, the virus could evade the innate immune response by causing delayed induction of proinflammatory cytokines and suppression of innate antiviral response in vitro. Second, MERS-CoV was able to infect key professional antigen-presenting cells that bridge the host innate and adaptive immune responses, namely, monocyte-derived macrophages and dendritic cells. The virus could utilize these antigen-presenting cells as vehicles for systemic dissemination. Third, MERSCoV could suppress the host adaptive immune response by infecting T lymphocytes and activating the extrinsic and intrinsic apoptosis pathways, thus leading to marked lymphopenia in severe infection. Finally, upregulation of Smad7 and FGF2 was identified to be an important mechanism involved in MERS-CoV-induced lung and kidney damage. Together, these novel findings helped to explain the unusually high virulence of MERS-CoV.