Team:Moscow City/Implementation

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Proposed implementation and Entrepreneurship
Overall Aim
To help horse owners start EHV treatment in a timely manner, by providing a rapid, sensitive and specific diagnostic test.
End Users
Our EHV test can be utilized to target both individual users (B2C scheme) as well as companies or authorities that will buy our test in bulk and distribute it. The rationale behind the two target groups is as follows:
Individual horse owners: farmers,
professional equestrians
Stables, Veterinarians
and Competitions Organizers

Horses are sick with something symptomatically similar to EHV. The owners can't figure out what it is and they require a quick answer to quarantine the horse and prevent the spread of the disease.

Stable managers need to identify the disease as soon as possible to prevent other horses from being infected.

Veterinarians need to perform diagnostic tests for several diseases as quickly as possible and with less equipment and reagents. For smaller facilities, on-site testing is not feasible, which increases the time required for the results to arrive.

Competition Organizers need to run the competition and prevent EHV from spreading to competing horses
Current Solutions
  1. PCR test that needs to be done in an authorized facility, which reduces turnaround time. Stables and individual owners do not have easy access to PCR tests or the cost is prohibitive. [1]
  2. Based on data from neighboring stables or farms and symptoms, individual owners determine whether or not it's EHV based on experience, which is not an accurate diagnosis and can easily lead to the horse’s death.
Why does our project matter?
Our goal is to show that animal care is an area worthy of attention. The experience of Covid-19 showed us that even people were not ready to fight a highly contagious virus.

Helping horses as part of the animal world encompasses not only equestrian rides and pure enjoyment but also reciprocal protection for both horses and humanity. As a by-product, this kind of assistance enables people to achieve Sustainable Development Goals [2] when implementing them in practice.
In the long run, it would also help developed and developing agricultural countries, mainly because their economies are sufficiently dependent on horses.
But what if we assist solely "strict numbers." Why shall we concentrate on horses? The answer lies on the surface. The formula of the most developed countries, which advance their agricultural technologies without eliminating previous production methods, perfectly suits the horse issue. In 2017, the impact of horses on the American economy was estimated at $122 billion and provided 1.7 million jobs [3]. Making it total that there are over 9.2 million horses in America [4]. That's more than the number of New Yorkers' citizens, in general.
Why did we choose EHV?
Equine Herpes Virus can be easily confused with other diseases such as flu or arthritis. Moreover, the symptoms of flu and EHV-1,4 are indistinguishablу [5]. So, it’s inevitable to have proper diagnostics to get the right treatment.
Additionally, several EHV outbreaks ceased recent Equestrian competitions [6] in Europe with claiming the lives of 10 horses.

Well, since it's a virus, the issue seemed to be solved by vaccinating all horses. However, vaccines do not provide сomplete protection from the EHV and most contagious types (EHV-1 and EHV-4) can still affect a horse. Also, vaccination works only under herd immunity.
Why this technology?
CRISPR-Cas allows us to sufficiently reduce the cost and the time (from 3 days to 3 hours) of the test. Moreover, 2 Cas-proteins protect us against the false-positive results, increasing our specificity.

Furthermore, there is a recent example of a CRISPR test approved for emergency use by the government. [7]
Based on the market needs for detection of EHV and other diseases in horses, we designed a rapid diagnostic test according to the following pillars:
  1. Point-of-care: (cell-free system, ability to use without equipment)
  2. Rapid results: (lactamase, etc)
  3. Specificity: (dCas13, two gRNAs etc)
  4. Affordability: It costs less than a PCR test since it requires less equipment and can be installed in more locations. Scale-up will also likely reduce reagent costs.
  5. Modularity: (ability to use multiplex approach to target a group of important horse diseases, in a single diagnostic kit)
  6. Easy-to-use: Our test does not require any blood samples, only a nasopharyngeal swab. Thus, it sufficiently reduces both the risk of harming the animal and reduces the time needed for more complex sample collection.
A real-world example
Let's imagine a situation where you decide to ride your favorite horse and come to the stable. The horse looks weak, doesn't want to eat, and has a watery nasal discharge and cough. Additionally, your hostler tells you that there has been an EHV outbreak at a nearby stable. You immediately call the vet; he or she comes and takes a nasopharyngeal swab from your horse.

But what will they do with it after coming to the lab?
  1. Extract RNA using HUDSON ( Heating Unextracted Diagnostic Samples to Obliterate Nucleases) (20 min)
  2. Do a reverse transcription and LAMP. (20 min)
  3. Does a T7 transcription. (20 min)
  4. Vet takes our paired ribonucleoproteins dCas13a/gRNA and loads them on the plate with the sample and control without the virus (40 min)
  5. Recognition: Add nitrocefin. If the color of the sample changes to red, the viral RNA is present in the sample. If the color of the sample and control are the same then viral RNA isn't present(10 minutes)
Result: if the well's color is red, it has an EHV there, so the horse is sick.
If the well's color has not changed or is the same as the positive control, it has no EHV.

Next step: the vet calls you and tells you positive or negative news.
Our Challenges
Raising funding for creating many kits of our system.
We need to produce test systems to sell them because you can't get far with a couple of samples. So the question arises: how to outperform it? The answer is simple: you need to have money.
How will we do it?
Many university accelerators and foundations in Russia give grants to support students and industrial projects and accompany their projects. Many of them are given even at the very "idea" stage. As soon as we test our sample on the virus, together with them, we will launch a small production, with which we will enter the Moscow stables.

Price of our test: it should be affordable not only for professional equestrians but for small farmers. 

How are we about to calculate it on the seed and early stages?

1. Calculate the cost price: cost of goods sold + OPEX (rent of equipment, utilities, salaries, etc.). CAPEX should be relatively small since all of our long-time ( longer than a year) assets are rented. We will meet CAPEX if our project succeeds and survive the early stage, but that's another story. [8-9]

2. Analyze our key competitors' test systems (mainly PCR and ELISA) in Russia and the rest world. Sure, we are scarcely able to cost more than them. However, since our test is faster and does not require expensive equipment, we hope to achieve it.

3. Do not forget to pay your taxes!
    And add them to the final price.

4. Consider Return on Investments for approximately 15-20% and keep in mind business development (after graduation, the University lab will say bye-bye, so we need to move somewhere). [10]

5. Get the final price.

Neither test based on CRISPR/Cas system is officially registered.

All the diagnostic protocols say that the gold standard of testing is PCR or antibody test. Moreover, it happens for human testing, while for the animal field, innovations often come later.

Perhaps, due to COVID, people will pay attention to express testing systems, and this situation will change. For example, in May 2020 the FDA emergency-use authority approved the CRISPR-based test for the coronavirus [7], so nowadays things are totally in favor of novel diagnostic technologies.

How will other scientists use our project?
First of all, scientists who will make their test system can take a look at our comparison of the existing methods of rapid diagnostics and choose the appropriate one.
They will also need to do DNA cloning, and for this, they will first have to do it in Snapgene or UGene. We have developed special tutorials for this.

Of course, they can read the whole lab part of our Wiki and use the protocols we gave there if that helps.

Download them:
Project Safety
After SARS-CoV-2 [11], many think that EHV can infect humans because humans have a herpes virus too [12]. But it's impossible, and neither EHV type can transmit to humans since EHV is species-specific. We additionally confirmed this question with Wright-Markey Professor of Equine Infectious Diseases Dr. Lutz S. Goehring.

In the early stages, our test requires LAMP so that experts will conduct it. They are also qualified to swab animals so that animals will not be harmed as well.

The swabs for the test are taken from the nasopharynx, so we do not harm the animal and do not take any blood from it.
Future of our project
First of all, We want to make sure that with better and cheaper diagnostics, the facts of outbreaks are better recorded. Since we know that unscrupulous horse owners sometimes hide rhinopneumonia in their horses. We are concerned that this will not cause additional outbreaks.

There are two ways of improving our project:
  1. Expand our test system to other viruses from the same family, such as Bovine alphaherpesvirus 1 (BoHV-1) or Human alphaherpesvirus 3 (HHV-3)
  2. Do experiments on reducing the number of samples and steps required for a reliable result, to minimize both cost and time needed for results.