INTRODUCTION

According to the United Nations Brundtland Commission, sustainability is defined as “meeting the needs of the present without compromising on the ability of future generations to meet their own needs” [1]. The UN Sustainable Development Goals(SDGs) serves as a worldwide framework for governments, agencies, companies and the general public to collaboratively upkeep or improve the quality of life for future generations. Our approach to sustainability included identifying and exploring how this biosensing system interacted with the SDGs, as well as interviewing experts in relevant fields to get feedback on the same. Due to the biosensor's versatility and its application in multiple fields, we also aligned its potential markets with the entrepreneurship business plan and focussed specifically on the immediate viral diagnostics market and a secondary environmental biosensing market. This report explores how our RNA-biosensor positively and negatively impacted three of the seventeen UN SDGs:

1. SDG 3 (Good health and wellbeing)
2. SDG 14 (Life below water)
3. SDG 15 (Life on land)

SDG 15: LIFE ON LAND

The Avian Influenza virus, commonly known as the Bird Flu, has infected millions of livestock for decades [2]. Recently, it has been causing severe infections with high mortality rates in poultry [3]. It has also been slowly transitioning to other animals such as domestic chickens, turkeys, quails, wild birds, and more [4]. Therefore, it is of great importance to study how this virus impacts land animals. Using this as our proof-of-concept, we explored how this virus affects forest and water ecosystems. Therefore, this can be connected to SDG 15: Life on Land.

Problem identifications:

There are many challenges associated with veterinary diagnostics when it comes to viral detection. Most farms and forests are in remote areas, far away from laboratories that perform diagnostic tests. There has also been little development in new detection technologies, especially those suited to detect viral pathogens in field settings. Current lab testing methods include RT-PCR, virus isolation, electron microscopy, and antigen enzyme-linked immunoassays (ELISA) [5]. These techniques are laborious, time-consuming, expensive or limited as a platform [5]. Another major issue in the field is that sample collection and transportation have an impact on viral detection as sampling and storage methods have been known to interfere with the compounds measured for identification (nucleic acids, surface antigens, etc.) [6]. These have been further explored below.

Problem 1: Sampling handling and transportation impact viral RNA integrity

To maintain the integrity of nucleic acids within the virus being tested, samples must be collected and transported with care; while on the field and once brought to the laboratory. Sample handling and transport plays a significant role in the properties of a viral sample, which include (1) swab tip material, (2) viral transport media (VTM), (3) media volume, (4) temperature of storage, (5) duration of storage, (6) inactivation of viral particles, (7) lyophilization [6] [7] [8].

Swab material is important as different materials interact with cells and cell-related particles differently - for example, the CDC recommends not using cotton and wood shafted swabs as it absorbs elution buffer, making it difficult to elute the virus [9]. The composition and volume of VTM, as well as the storage temperature and duration determine the stability of viral capsids and internal RNA [6] [10] [11]. Viral inactivation makes the transportation of the virus safer, but some of their methods that physically inactivate (heat, UV) the virus can impact its RNA stability [12]. Lyophilization allows for the transport of the virus over longer time periods, but improper cooling rates and absence of cryoprotectants may lead to degraded virions and viral RNA [13] [14].

Laboratory processes used to extract the virus from its sample solution may also impact the detection sensitivity, such as (1) thawing rate (for lyophilized samples only). Slow thawing rates create ice crystals which penetrate viral capsids and therefore, must be increased to preserve the virion [14]. Aside from this technique, many other methods used for viral diagnostics have low yields that have low sensitivity as a result [15].

Solution to problem 1:

Our RNA biosensor addresses the issue of sample storage and transportation methods, which have an impact in virion stability and RNA integrity. Rather than having chemical storage methods or freeze-drying samples, we have designed a simple buffer system that will inactivate and degrade the viral capsid while maintaining internal RNA stability. This eliminates the need for transportation to a lab, or bringing VTM and other toxic chemicals to a field-setting. Therefore, this simplifies the diagnostic testing process remarkably by providing rapid results on the field.

Problem 2: Current detection methods are not scalable during outbreaks

There are many aspects of veterinary diagnostics that are disadvantageous in the case of a viral outbreak. One of these include lack of equipped labs for testing in remote areas [16]. Since there aren't many labs that would offer viral laboratory diagnostics in a given area, wait times are likely to be high for such requests. Viral identification methods (except for antigen tests) involve complex procedures, sophisticated technology, and toxic chemicals [5]. Additionally, most veterinarians presently collect samples from the field, store them in a VTM and have them transported to a nearby laboratory that can perform the required tests. Sample storage and transportation can also result in low diagnostic accuracy, as outlined in problem statement 1. Moreover, there are viral strains such as the Highly Pathogenic form of the Avian Influenza virus, for which research can only be done in BSL 3 or BSL 4 labs [17]. Molecular techniques such as PCR may be performed in a BSL 2 lab after viral inactivation, which can be correlated with viral RNA degradation [12]. All these issues lead to a singular conclusion: there aren't enough laboratories for detection of veterinary viruses, and the distribution of the current ones is not uniform. This makes global viral testing of animals during a severe outbreak laborious and complex.

Moreover, these laboratory tests are expensive [5], which may incentivize farm owners to conduct tests on only the severely-ill or symptomatic cattle. This may reduce the quality of care provided to the animals, and allows soon-to-be-symptomatic poultry to go unnoticed until it's too late. To summarize, viral diagnostics are developed to some extent for low testing periods, but may turn into a significant challenge when testing rates increase. Therefore, these issues must be addressed with realistic solutions, to make veterinary authorities more prepared in the unfortunate case of a viral outbreak.

Solution to problem 2:

Our test-kit also addresses the issue of lab scarcity in remote areas like forests or mountains. Being a user-friendly, cheap, and real-time biosensor, this eliminates the need to take viral samples to a laboratory - which provide faster results and is more convenient than depositing samples at nearby labs. The cost-friendliness of the kit may also encourage farmers to conduct more routine testing that catches early infections in time, and benefits the farmer over the long-term.

Long term social, environmental, and economic impacts:

This biosensor would have a significant impact in veterinary diagnostics, as it will make the process of mass viral testing easier, faster, more convenient, and field-deployable. It will relieve pressure off labs that conduct such testing, and allow them to develop a vaccine for such transmissible and pathogenic diseases. Environmentally, it is a simpler technology than most other detection methods, and therefore generates less amounts of toxic chemicals that go into ecosystems. Finally, it will have an immense economic impact as it will contribute to the early detection of the virus, which is associated with lower mortality rates. With a restoration in egg production, the poultry industry will see a decrease in trade losses.

We had the privilege of speaking with a central stakeholder: Dr. Mohammed Careem - a professor and expert in veterinary medicine, with experience working with Avian Influenza. After speaking with Dr. Careem, it was clear that we had selected the right virus for a proof-of-concept. He elucidated that a key industry concern presently is the lethality of Avian Influenza, with infected birds dying within four days. However, he had some concerns regarding the method of testing, and suggested that we environmentally monitor the pathogen rather than testing in poultry animals, as that is an external method of transmission due to wild bird migration patterns. He also suggested that we connect with more stakeholders in the industry who are involved in meat production, rather than egg production.

Positive/negative interactions with other SDGs:

This positively impacts SDG 8, as this particular virus has cost the US economy $1.4 billion in decreased egg production due to high mortality rates. Restoring this economy to its previous state will allow for the reallocation of financial resources towards other matters of high economic importance. It also has a positive impact on SDG 12 and 13, as this would result in significantly less vehicle usage (due to elimination of sample transportation steps), which will result in lesser energy usage in the form of fossil fuels, impacting global warming. It negatively impacts SDG 3, as the person conducting the test will potentially spend less time travelling to nearby laboratories and more time in the environment where the pathogenic virus being tested is present. Therefore, this poses an increased risk to the health of the individual performing the tests. It also negatively impacts an aspect of SDG 8, as the reliance on laboratories would decrease, which may result in lab technicians losing their jobs.

SDG 3: GOOD HEALTH AND WELLBEING

This year's project was centered around a portable, user-friendly RNA biosensing kit that can be used to detect any given virus of a known sequence. While it was mainly designed for testing nasal samples in poultry, it has massive potential to be used in the unfortunate case of a viral outbreak, along with less communicable viral infections. Therefore, it directly applies to an important SDG, Good Health and Wellbeing.

Problem identifications:

The purpose of this test-kit is to reduce the need for dependency on Polymerase Chain Reactions (PCR) for viral identification, as PCR tests are expensive, time-consuming, and require fancy lab equipment and reagents to be performed [21]. Probably due to such restraints, along with some other socioeconomic factors, low testing was observed during the COVID-19 pandemic in a multitude of countries, as outlined in Wei et al. (2020). It also correlated low testing with high mortality rates, which has been summarized in the figure below [18].

Problem 1: Low testing rates for viral infections

There can be many possible explanations for low viral testing rates. Some of them may be directly connected to the costs and time requirements of a PCR reaction (80-100$/test [26]) or antibody test(~50$/test [26]) - those belonging to low socioeconomic classes may not want to risk spending their money on the test if they have a mere cold [19]. Others may be due to issues in the public health system, such as insufficient funding, lack of adequate laboratory capacity and the number of medical professionals who can administer and perform the tests [20] [21]. There can also be regulatory delays, and unequal distribution and access of materials and reagents [21] [22]. Finally, there may be socioeconomic factors at play - people may not be educated on the importance of viral testing, especially in the case of an outbreak [23].

Due to the versatility of the system, this biosensor could also potentially be used to test bodily fluid samples for sexually transmitted diseases (STDs). In this context, the social stigma associated with having an STD (for example, the Human Immunodeficiency Virus) may prevent someone from going to their local healthcare provider and getting tested, despite the importance of early detection in improved patient outcomes [23].

To summarize, low testing is directly associated with high mortality rates, especially in the case of a viral outbreak. Low testing rates may be because of the testing method itself, faults in public health care infrastructures, and socioeconomic factors. With respect to sexually transmitted diseases, low testing may also be related to social stigmatization of the disease. Finding a solution to these issues is necessary, as in the case of serious viral infections, early intervention upon identification can significantly impact lives.

Solution to problem 1:

Current viral testing methods (PCR tests, antigen and antibody tests, etc.) yield low viral rates due to many economic, social and issues in the public health infrastructure. While our test-kit does not address all these issues, it significantly reduces some of the challenges. These include (a) decreasing the price per test to 7.67$, (b) inadequate laboratory capacities and available healthcare practitioners to perform the test and (c) providing populations with stigmatized viral diseases access to testing methods without visiting a medical facility. Therefore, this test-kit would increase viral testing rates, directly impacting SDG 3.3 and 3.9.

Problem 2: Diagnostic methods for viral infections contribute to community transmission

There may be a multitude of reasons why the public may not get tested if they have a viral infection. However, another important consideration is the nature of viral testing itself: the approved diagnostic method worldwide is RT-PCR testing, which requires the patient to travel to their nearest medical facility and let a health practitioner collect a nasal swab sample. This fundamentally contributes to the spread of the virus as it compels the possibly infected individual to travel to their nearest medical facility, while spreading the virus through aerosol droplets and surface contamination [24].

Public health agencies have tried to measure and control this phenomenon with contact tracing, where they try to map the group of people a given infected individual interacted with [24]. They use this data to inform potential asymptomatic or presymptomatic carriers to self-isolate and prevent spreading the infection [24]. Ferretti et al. outlines how mathematical models predicted that digital contact tracing had the potential to stop the COVID-19 pandemic entirely [24]. Some research also suggests that at-home antigen test kits could also be used to reduce community transmission, as it allows for isolated testing without going to a medical facility [25]. These measures underline the necessity and importance of at-home, isolated viral testing that eradicates the need to go to a medical clinic to get diagnosed.

Solution to problem 2:

Fundamentally, viral testing contributes to the spread of the communicable virus of concern. Our biosensor allows for at-home testing in the unfortunate case of a viral outbreak, where isolation and social distancing is crucial. The user friendliness, sustainable design, and low cost make this a very good choice for at-home, real-time testing if an individual suspects they have a viral infection that they can spread to others. This will prevent community transmission, as a sick person would only be required to go to the hospital if the severity of the symptoms worsens, rather than for a diagnostic test. On a population level, this will allow public health agencies to be better prepared and control the spread of a virus during the next epidemic.

Long term social, environmental, and economic impacts:

This biosensing system will increase viral testing rates, especially in developing countries where test costs limit rates. This will in turn reduce community transmission of future viral outbreaks and improve contact tracing measures - both of which will contribute to the improved outcomes of the outbreak, along with ameliorated emergency-preparedness of public health authorities. It would also allow researchers to spend more money and resources on developing vaccines for such pathogens, rather than focussing on virus-specific detection such as antigen and antibody testing.

This would have a combined impact on the healthcare economy, as it would allow for the reallocation of financial resources to address more pertinent challenges in the public health sector. It would also reduce the environmental waste generation associated with PCR and antigen tests, which would leach lesser amounts of harmful chemicals into the environment.

We had the opportunity to discuss SDG 3 with Dr. Alyson Kelvin, a virologist and vaccinologist who has conducted research in Avian Influenza, SARS-CoV-2, and the Monkeypox viruses. She suggested that although larger, federal governments may already have the resources and money they need for adequate testing, there is potential in selling this kit to smaller municipal governments. Remote areas, for example small towns with minimal lab infrastructure would benefit from a product like ours. She also believes that there is a possibility that the highly pathogenic Avian Influenza mutates and becomes transmissible for humans and therefore is an important viral target to consider.

Positive/negative interactions with other SDGs:

This platform positively impacts SDG 8 (Decent Work and Economic Growth) and SDG 12 (Responsible Consumption and Production). PCR tests cost 80-100$, while antigen and antibody testing costs approximately 50$. Since our test-kit is at least 7 times lower in cost, it will save institutions and governments copious amounts of money that they spend on PCR and antigen tests, which will allow it to be reallocated towards other issues. It will also allow savings in the form of wages as this kit does not require any laboratory personnel and can indicate the presence or absence of a virus directly to the user from a nasal sample. It also positively impacts SDG 12, as the kit does not involve any cold chain logistics that lowers energy usage per kit; and uses minimal amounts of plastics and chemicals that prevents large waste volumes. However, it will also negatively interact with SDG 8, as it will cause a subset of laboratory technicians who majorly run viral diagnostics in medical facilities to be unemployed.

SDG 14: LIFE BELOW WATER

Although our system was primarily designed for veterinary diagnostics, it shows incredible potential to be used in environmental monitoring applications. The Earth's natural water bodies have diverse ecosystems, most notably comprising of fish and other marine animals; and a multitude of bacterial, algal, and viral species [27] [29]. Research suggests that monitoring viral concentrations in water bodies is beneficial as it is an indicator of many oceanic processes such as eutrophication, acidification, along with reflecting the health of eukaryotic marine organisms [27]. Although our test-kit is primarily catered toward poultry testing in farms, it shows significant potential in environmental monitoring of prokaryotic and eukaryotic marine organisms. It is also worth noting that Avian Influenza spreads through wild birds (waterfowls in particular) via ponds, lakes and rivers in Canada, which makes viral detection in natural water bodies crucial in remote areas [36].

Problem identifications:

Viruses have been known to cause significant impacts in natural water quality, including ocean ecosystems and their biochemical compounds [27]. Bacteriophages, which are the dominant class of viruses in the ocean, infect many types of microorganisms and participate in viral shunting - where they release their organic matter which either accumulates or gets digested by other microbes [29]. This process plays a role in releasing nitrogen and phosphorus into natural water bodies, which contributes to ocean acidification and eutrophication [28].

They also cause viral lysis in marine algae, which plays a vital role in regulating carbon dioxide and oxygen levels through photosynthesis [28]. Thereby, they can also have positive impacts in marine ecosystems by regulating the number of algal blooms and thereby ocean acidification [27]. Moreover, research shows that there is unexplored potential in viruses to enhance carbon sequestration efficiency in the oceans [27].

Lastly, there are a range of viruses which infect fish - they enter the integumental tissues such as the gills and fins, and go on to induce viral lysis in different body tissues that ultimately kill the host [31]. Infected fish release more viral particles by shedding through feces, urine, mucosal and gill secretions, and gametes [32]. These particles further go on to infect more viruses exponentially, and therefore must be monitored to maintain good marine health in water ecosystems. Viruses infect a variety of fish, which (1) deteriorates their appearance (important in ornamental fish), (2) causes mild, non-notifiable infections, and (3) causes acute, notifiable infections with high mortality rates [33]. Current testing methods include taking affected fish to a lab, and carrying out PCR reactions, virus isolation and identification, necroscopic analysis [34].

Creating a cheap, field-deployable testing system that addresses all these issues will allow for increased testing for such a prevalent issue. Moreover, research and development towards vaccines or antimicrobials will be greatly benefited if there is an easier way to test for these viruses in these contaminated bodies of waters.

Problem 1: Viruses destroy photosynthetic bacteria and algae that contribute to eutrophication and ocean acidification

Viruses have been known to cause bacterial mortality in 10-20% of heterotrophic bacterial species and 5-10% of autotrophic bacterial species [27]. For example, cyanophages infect some picoeukaryotes and photosynthetic cyanobacteria - the Synechococcus and Prochlorococcus genera in particular [27]. They also infect a range of algae - from unicellular microalgae to complex macroalgae [27].

Viral lysis releases nitrogen and phosphorus, which are stored in nucleic acids and amino acids, along with reducing the number of microbes involved in carbon fixation [28]. This is most notably measured through the dissolved organic content for each of these elements. Shiah et al. suggests that viral lysis could be attributed to 145 gigatons of carbon and 27.6 gigatons of nitrogen per year. This organic matter can be classified into (1) particulate organic matter (POM) which does not get recycled easily and (2) dissolved organic matter (DOM) which is easier to break down and be incorporated into other bacterial biomasses [29] [27].

This directly decreases the rate of carbon fixation and thereby increases the total dissolved carbon in water bodies [28]. Therefore, some virus strains contribute to ocean acidification and need to be monitored to know which water bodies viral particle concentrations need to be reduced from. Current microbiological laboratory methods to measure the amount of virus in a natural water sample include Transmission Electron Microscopy (TEM), flow cytometry, epifluorescence microscopy and denaturing gradient gel electrophoresis (DGGE), among others [30].

Problem 2: Viral infections in marine animals

There are multiple viruses that cause infection in a variety of fish species. Multiple strains of viruses infect a variety of fish, which (1) deteriorates their appearance (important in ornamental fish), (2) causes mild, non-notifiable infections, and (3) causes acute, notifiable infections with high mortality rates [33]. Most mortality rates range from 80-100%, and those that survive these infections have a high chance of being carriers and usually need to be isolated [34]. An overview of such viruses has been outlined in Table 2, which underscores the severity of the infection.

Table 2. Overview of marine viruses that infect fish and cause high mortality rates

Virus strain	Fish species	Mortality rate
Cyprinid herpesvirus-1	Cyprinus carpio	80-100% [38]
Cyprinid herpesvirus-2	Carassius auratus	90-100% [37]
Cyprinid herpesvirus-3	Cyprinus carpio Carassius auratus (carrier) Ctenopharyngodon Idella (carrier)	90-100% [37]
Novirhabdovirus	Perca flavescens Onchorhynchus mykiss Onchorhynchus tshawytscha	80-100% (for P. flavescens) [39] 50-100% (for O. mykiss and O. tshawytscha) [39]
Vesiculovirus	Rutilus kutum	10-85% [41]
Megalocytivirus	Pagrus major	20-100% [40]

Current testing methods include taking affected fish to a lab, and carrying out PCR reactions, virus isolation and identification, necroscopic analysis, which have a significant negative impact and have been discussed in the following section [34].

Solution to problems 1 and 2:

Viral monitoring in oceanic ecosystems holds significant value for aquatic veterinarians, geomicrobiology researchers, microbiologists, fishermen, etc. Current testing methods are accurate, but are expensive, time-consuming, and restricted to a laboratory setting. Our field-deployable, user-friendly biosensor can provide real-time results and costs only 7.87$. It has a simple sampling strategy where one extracts 2 mL of liquid, and adds it to the sensor box, and gets a positive or negative result in real-time. This will help researchers by saving transportation time of samples to a lab, simplify the process of monitoring fish and oceanic microbe health, and ultimately conduct more environmental viral testing while spending less money.

Long term social, environmental, and economic impacts:

The lab procedures that are used to currently test for viruses in natural water bodies are expensive, require highly technical lab equipment, and have high wait times for results (not to mention the resources the user spends on transportation of the samples). Our test-kit is relatively cheaper, user-friendly and convenient field-deployable biosensor that serves as a solution to these issues.

We had the opportunity to discuss SDG 14 with Christine O'Grady, the Executive Director of Advancing Canadian Water Assets (ACWA) - which is an Alberta-based research initiative that measures Sars-CoV-2 concentrations in wastewater as a method of contact tracing for remote areas. Her research team mainly collects water samples from the Pine Creek, and sends it to the University of Calgary's geomicrobiology lab to perform RT-PCR reactions to determine viral identity. She mentioned that there are many remote communities across Canada (for example, Yukon) where viral testing is limited due to the high shipping and handling costs. Moreover, we agreed upon the fact that even if shipping is possible, our test-kit would significantly ameliorate testing processes by providing real-time results in the field, which would make it commercially-viable.

Positive/negative interactions with other SDGs:

Designing this field-ready biosensor positively interacts with SDGs 2 (Zero Hunger), 13 (Climate Action), 8 (Decent Work and Economic Growth) and 12 (Responsible Consumption and Production). Environmental monitoring of viruses would lead to earlier detection of fish infections, which in turn would increase the number of surviving fish populations. Thereby, this contributes to increasing the available seafood for a given region and contributes to SDG 2. It also serves to manipulate the extent of ocean acidification (a metric of global warming), which positively contributes to SDG 13. It also positively impacts SDG 12, as the kit does not involve any cold chain logistics that lowers energy usage per kit; and uses minimal amounts of plastics and chemicals that prevents large waste volumes.

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