Overview of Project DR. SWITCH (Disease-associated RNA Switch)

Our project focus on developing an on-site subtypting method for Influenza A virus subtype H5N1 and H7N9 using toehold switches. To facilitate future toehold switch project, we also developed an online software program for designing toehold switch, and constructed toehold switch cloning tool that allow easy construction and validation of toehold switches.

Influenza A is a rapid changing disease that causes 5,000,000 of death annually worldwide. Among different subtypes, highly pathogenic avian influenza has the highest mortality rate. Challenges of disease control in the modern world with high population mobility remains at the speed and accuracy of diagnosis. However, nowadays influenza A subtyping method rely greatly on RT-PCR, which requires long time, expertise and laboratory space. Meanwhile, a novel type of riboswitch, namely toehold switch, shows its potential in subtyping Influenza A with quicker detection and lower production cost. By combining cell free system and toehold switch, a rapid on- site detection method for influenza A subtype H5N1 and H7N9 is designed and under investigation. It has high potential to be used widely in, but not limited to, animal farms, and border inspections and schools wherever expertise and laboratory equipment are not readily available.

We hope that our project can (i) provide rapid detection method with higher accuracy at a lower production cost; (ii) stop Influenza A pandemic by early on-site detection; and (iii) ease the stress on public health service when disease attacks, especially in less developed countries.

Influenza Type A

Influenza A can be spread rapidly throughout poultry flocks and cause severe illness, or even death in human. The most notorious pandemic was the “Spanish Flu” in 1918, which killed 50 million people worldwide (1). Influenza A virus poses large social and economic burden. Each year in the United States, it is estimated that around 600,000 lives and $90 billion US dollars are lost due to influenza A virus (2). Influenza A can be subtyped according to the types of hemagglutinin (HA) and neuraminidase (NA) on the virus surface (figure 1). Since there are 16 types of HA and 9 types of NA, there are 144 possible subtypes. Different influenza A subtypes possess different properties. For example, the mortality rate of infection by the subtypes H5N1 and H7N9 is much higher than that of H1N1.

Avian influenza

In this project, we aim to construct a set of artificial RNA biosensors to detect different influenza A viral genes, including the hemagglutinin and neuraminidase genes. We consulted local medical expert (link) and found that there is an urgent need for fast and on-site subtyping method for Avian influenza compared with other subtypes in Hong Kong. Avian influenza are influenza A viruses that adapted to birds, which can be classified into high pathogenicity (HP) or low pathogenicity (LP). Since Hong Kong is the stopover point of migrating birds, the chance of avian flu outbreak in Hong Kong is much higher than in other places. Together with the fact that Hong Kong is a highly populated city with great passenger throughput per day, any outbreak of avian flu in Hong Kong may easily cause pandemic.

Therefore, we focused on constructing biosensors for subtyping the notorious Avian influenza. Among the subtypes, subtype H5N1 and H7N9 are the most urgent subtype that require need method to diagnose.

H5N1: The notorious flu

H5N1 is the most notorious highly pathogenic avian influenza. The first epidemic outbreak of H5N1 in human happened in Hong Kong in 1997. The flu was then spread to the entire Asia. According to the World Health Organization, there was 630 confirmed human cases since 2003 which killed 375 people with a mortality rate of 60%. The disease not only create tremendous economic burden to the health care system, it also greatly impact the poultry industry. During the outbreak of H5N1 in Hong Kong, 3.5 million chicken was slaughtered. About $10 billion US dollars had lost due to H5N1 outbreak. Although the risk of H5N1 pandemic outbreak in human population is considered to be low recently, it is considered as endemic in poultry in six countries (Bangladesh, China, Egypt, India, Indonesia, and Vietnam).

H7N9: The next H5N1?

H7 virus was thought to be only circulated among avian hosts but human infection is recently reported. The first case of human infection was recorded in China in 2013 (Tanner, Toth & Gundlapalli, 2015). According to the World Health Organization (WHO), 1533 human infection cases were reported, with a mortality rate of 39% ("Monthly Risk Assessment Summary", 2017). In Hong Kong, 4 confirmed human cases were reported so far. H7N9 cased economic loss of about $6.5 billion in China (Horby, 2013). Among all the avian influenza virus, H7N9 virus was found to have the highest ability to infect humans and circulate in birds (Zaraket et al., 2015). WHO warned that the human infections are unusual and need to be carefully monitored. According to the Centers for Disease Control and Prevention (CDC) of the United States , H7N9 is the subtype that has the greatest potential to cause a pandemic in recent year compared with other subtypes. It is worried that H7N9 may cause next pandemic since the virus is evolving mechanism for human- to- human transmission (Morens, Taubenberger & Fauci, 2013).

The need for new subtyping method

To effectively control the possible outbreak of avian influenza, a simple and rapid on- site method is needed for detecting the virus in both human and poultry. However, nowadays on-site diagnostic method, such as Rapid Influenza Diagnostic Tests (RIDTs), can only identify the influenza A virus but cannot subtype it (3). Traditional influenza A subtyping method rely on qRT-PCR (Daum et al., 2007). Although the technique is highly sensitive and specific (Tsushima et al., 2015) , it is not suitable to be relied on during the spread of disease, since it requires long time, and cannot perform in poor condition where expensive equipment and technical expertise are not available. Failure of immediate respond to the spread of disease may result in pandemic (Ross, Crowe & Tyndall, 2015). Meanwhile, a novel type of riboswitch, namely toehold switch, shows its potential in detecting viral RNA on- site with short detection time and low production cost.

RNA toehold switches

The artificial RNA biosensors we used is called toehold switch, which is first developed and published in 2014 by Green et al (5). It is a motif in mRNA that allows the translation of downstream protein coding sequence when a specific trigger RNA binds to it. The trigger RNA binds to the switch region of the toehold linearizes toehold secondary structure. This then releases the Ribosomal Bing Site (RBS) from the loop, allowing a ribosome to bind on it. The ribosome can then read along the coding region of the toehold switch, hence giving off a signal.

Cell free system

The toehold switch technology will further apply in cell free system to allow on-site detection for influenza A in. It has a high potential to be used widely in, but not limited to animal farms and border inspections wherever expertise and laboratory equipment are not readily available. Recent findings showed that toehold switches can be used to detect Zika virus and Ebola virus (6, 7). However, to the best of our knowledge, none of the up- to- date research has shown that toehold switch can be used to subtype Influenza A while existing technology shows limited advantages on Influenza A diagnosis.

Altogether, the project aims to apply toehold switch technology in influenza A subtyping and offer convenient tool to facilitate the design of toehold switch. It is hoped that our method could suppress the spread of pathogenic Influenza in a timely manner.