CENSAM Research Initiative Project MIT PI: Prof. Andrew Whittle Norovirus Aptasensor Masaaki Kitajima MIT PI: Prof. Andrew Whittle Collaborators: Prof. Jianmin Miao, Mr. Nan Wang (SMA) NEC Solution Innovator Co. Ltd.

Cruise with Norovirus…? 570-800 Deaths 56,000-71,000 Hospitalizations 400,000 Emergency department visits 1.7-1.9 million Outpatient visits 19-21 million Total illnesses (~5 illness episodes in a person’s lifetime) Major cause of acute gastroenteritis Food- & water-borne Diarrhea Vomiting Stomach ache Fever etc. Norovirus is a major cause of foodborne and waterborne acute gastroenteritis with various symptoms often reported as stomach flu. As you can see in this figure showing the estimated annual burden of norovirus in the US, it causes various disease outcomes from even deaths to unreported illnesses, a total of 19-21 million illnesses per year. In other words, it is estimated that a US resident would experience 5 episodes of norovirus gastroenteritis per lifetime. By the way, this virus is notoriously known for cruise ship outbreaks, and many people have had a bad experience cruising with norovirus. Estimated Annual Burden of Norovirus in US (data from Hall et al., 2013)

Norovirus in Environmental Waters USEPA Contaminant Candidate List Highly persistent Small size (~38 nm) ~105 virus/L1) ~106.5 virus/L1) ~105 virus/L2) Norovirus is also known as an environmental contaminant. In fact, it is included in the USEPA Contaminant Candidate List, which lists emerging contaminants known to occur in public water systems. It is difficult to control environmental contamination of norovirus because of its high environmental persistence and small particle size. Norovirus has been found at a few points in Singapore’s urban water cycle, although there has been no reported evidence on the presence of norovirus in potable water. According to the published reports from Singapore, norovirus was found to occur in untreated wastewater with a concentration of up to 10^6.5 virus per liter and in treated wastewater and surface water with up to 10^5 virus per liter. Reports from Singapore Aw & Gin, 2010 No evidence Liang et al., 2015 (Source: National Climate Change Secretariat, Singapore)

Existing Detection Methods vs Aptasensor Polymerase Chain Reaction (PCR) Aptasensor (aptamer-based biosensor) Recognition element Transducer Biosensor ⇨Aptamer ️️⇨Electrochemistry RNA Aptamer Synthetic DNA/RNA Fold into 3D conformations binding a specific target Viral protein (Modified from Beier et al., 2014) RNA extraction (~1 h) These results have been obtained using quantitative polymerase chain reaction, which is most commonly used for detection of pathogens in the environment, but it takes at least several hours to obtain the results and has limited on-site application. One possible way to achieve real-time on-site monitoring is the use of biosensor, which rapidly converts a biological response into an electrical signal. We use aptamer as a recognition element and electrochemistry as a means of transduction. Aptamer is a synthetic DNA or RNA that folds into unique 3-dimentional conformations binding a specific target. Reverse transcription-PCR (~3 h)

Advantages of Aptamer as a Recognition Element of Biosensor High affinity Comparable to / higher than antigen-antibody affinities High specificity Recognize unique surface structure of the target molecule High thermostability Robust against ambient temperature variation Reusability Easily regenerated by reversible heat denaturation Production via in vitro chemical synthesis High purity and constant quality with low cost Easy modification Immobilization on surfaces Viral protein And aptamer offers various advantages as a recognition element of biosensor. The binding affinity is comparable to or even higher than antigen-antibody affinities. It is highly specific because it recognizes unique surface structure of the target molecule. Its highly thermostability makes it robust against ambient temperature variation and reusable with regeneration by reversible heat denaturation. It can be produced via in vitro chemical synthesis, which provides high purity and constant quality with low production cost. Lastly, we can easily attach chemical modification to an aptamer for various purposes such as immobilization on surfaces. Aptamer

Fabrication of a Prototype Aptasensor Model Virus: Mouse Norovirus Easily propagated in vitro Similar to human norovirus Not pathogenic to humans Gold electrode 2. Electrochemical Measurements 1. Aptamer Immobilization S Thiolated aptamer Self-assemble/immobilization Mouse Human We are currently working on fabrication of a prototype aptasensor and virus propagation to obtain a large amount of virus particles for seeding experiments. Mouse norovirus, the virus we are using for our experiment, is a good model of human norovirus because it is structurally similar to human norovirus but not pathogenic to humans, so it is safer to handle in the lab. We are about to start working on aptamer immobilization on the surface of gold electrode and electrochemical measurements. Ms Kalaivani Mani (RA) working on virus propagation

Application of Norovirus Aptasensor Real-time, on-site environmental monitoring Viable norovirus Damaged (Non-viable) Viability discrimination The goal of this project is to develop a portable biosensor for real-time on-site monitoring of norovirus in the environment. One of the attractive features of aptasensor is the potential capability of viability discrimination – in other words, selective detection of viable virus particles. Since aptamer recognizes surface structure of viral coat protein, we can expect aptamer not to capture a damaged virus particle which is non-viable and irrelevant to public health risks. This aptasensor technology can also be applicable to monitoring of other pathogens by simply replacing the norovirus aptamer with another pathogen-specific aptamer. Thank you for your attention and I would be happy to take questions over coffee. Application to other pathogens Masaaki Kitajima (kitajima@smart.mit.edu)