iGEM Athens 2018
MERS-CoV (Middle- East Respiratory Syndrome Coronavirus) is a Coronavirus, first identified in the Kingdom of Saudi Arabia in June 2012. Since then, the World Health Organization (WHO) has reported 2200 laboratory-confirmed MERS-CoV cases with 790 associated deaths in 27 countries. It is transmitted through human-to-human contact, as well as through direct or indirect contact with host animals, mostly camels. The virus attacks the human respiratory system. The most common signs and symptoms associated with MERS-CoV at presentation are fever, cough, and shortness of breath. Typically presenting symptoms include fever, cough, chills, dyspnea, myalgia, abdominal pain, nausea, vomiting, and diarrhea.
Why is MERS-CoV a threat?
MERS-CoV is highly pathogenic and causes a series of non-specific symptoms which complicate the diagnosis. Some laboratory-confirmed cases of MERS-CoV infection are reported as asymptomatic, unknowingly spreading the disease. A key fact is that the mortality rate of humans infected by MERS-CoV is above 35%. Outbreaks caused by MERS-CoV have occurred in several countries, with the most significant ones seen in Saudi Arabia, United Arab Emirates, and the Republic of Korea. Saudi Arabia and the United Arab Emirates are countries within the geographic range where the virus is endemic. However, the epidemic in the Republic of Korea proves that the virus is a global threat. The expansion and easy access of modern transport networks, apart from the apparent positive societal feedback it may bring, it also facilitates global pandemics of communicable diseases. As a result, the WHO has declared MERS-CoV as one of the most likely to cause a future epidemic and urges for further research.
How can we prevent a future MERS-CoV epidemic?
Despite public awareness and implementation of public health care measures, the number of laboratory-confirmed cases of MERS-CoV is still rising. Therefore, it is clear that the development of an easily accessible and rapid viral diagnosis procedure is necessary. Our goal is to develop a molecular diagnostic kit for the detection of MERS-CoV. Trying to meet the existing societal needs, we are aiming for an easy-to-use, rapid test that is reliable and safe, can be used on the field, even by the untrained, as opposed to existing methods of diagnosis.
Our biosensor is based on the Toehold-Switch technology. Toehold switches are hairpin-shaped riboregulators that precede a protein coding sequence in a synthetic mRNA molecule. The conformation of the switch regulates the expression of the protein; in the absence of a trigger complementary RNA sequence, the switch region folds, inhibiting the binding of the ribosome to the RNA and subsequently the expression of the coding sequence. The synthesis of the protein is allowed only if the target sequence is present and binds to the switch region, causing its unfolding. This protein acts as the reporter of the system, catalyzing a reaction which causes a visible signal output.
How does the MERS-CoV diagnostic kit work?
The toehold switches designed for our biosensor will be activated by characteristic sequences of the MERS-CoV genome and regulate the expression of an engineered enzyme. We will evaluate the potential of using a bacterial trehalose hydrolase enzyme (trehalase) as the reporter protein. The enzyme hydrolyses trehalose, a glucose dimer, leading to glucose accumulation in the sample. The produced glucose can then be easily detected by a standard glucose assay, such as a routine Glucotest strip and be directly correlated to the presence of the virus. Incorporating this mechanism as a DNA construct in a cell-free transcription and translation system, creates a robust, genetically engineered circuit that can be used as a biosensor.
Complementary to the diagnostics kit, our team also aims to create a set of bioinformatics tools that take into consideration the requested viral genome and the sample origin, suggesting the best candidate target sequences to be examined. On this scope, we are designing Pre.Di.C.T. (Predictive Diagnostic Custom Toehold-Switch), a user-friendly workflow that will facilitate the design of molecular-diagnostics systems for future research.
- Corman et al., (2014), “Performance and clinical validation of the RealStar ® MERS-CoV Kit for detection of Middle East respiratory syndrome coronavirus RNA”, Journal of Clinical Virology 60(2): 168-171
- Drikic et al., (2018), “Split trehalase as a versatile reporter for a wide range of biological analytes”, Biotechnology and Bioengineering 155(5): 1128--1136
- Pardee et al., (2014), “Paper-Based Synthetic Gene Networks”, Cell 159(4): 940–954
- Widagdo et al., (2017), “MERS-coronavirus: From discovery to intervention”, One Health 3: 11-16