Team:Moscow City/Human Practices

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Environmental, social, moral, scientific values
We innovate in the agricultural-animal diagnostics sector
The latest developments in synthetic biology more often affect those areas that are directly related to human health.

Using the example of horse breeding, we found out that diagnostic methods, vaccines, medicines were often invented in the 20th century and have not been updated since.

Of course, groups of scientists conduct their research in these areas, produce valuable articles, but it only comes to the practical use of their results if it becomes economically justified. Unfortunately, it often becomes economically justified after significant incidents like COVID-19.
We want to show that animal care is an area worth your attention. The experience of Covid-19 showed us that even people were not ready to fight a highly contagious virus.
The fight against Covid-19 [3] has led to the rapid development of virus research, diagnostics, vaccination, and preventive measures
An example of this is the outbreak of the equine herpes virus in Valencia this year. Than EHV-1 cases – unrelated to the outbreak in Spain – have been reported in Italy, Sweden, Great Britain, Ireland and France [1].

The most reliable way to avoid an outbreak is to follow preventive measures, isolate the sick horse in time. This is precisely the goal of our project - to create a test for rapid diagnosis, allowing the detection of an infected horse in a short time, without additional equipment.

CRISPR-Cas allows for the diagnosis of diseases without expensive equipment and specially trained personnel. They are suitable to prevent outbreaks and timely isolation since they can be used in the field [2].

CRISPR-Cas methods have just begun to develop and apply rapidly, there is not enough research on their application, and with our work, we contribute to the study of Сas13.

We also actively followed publications and public lectures by professor Konstantin Severinov, a Russian authority on CRISPR-Cas technologies. We consulted him about the use of Cas13, the research groups studying this protein in Russia. He inspired us to work with RNA-guided Cas13, especially in the COVID-19 pandemic caused by the RNA virus.

When we started our work, we set ourselves the goal of creating a test for rapid diagnosis

The ideal test that we are also aiming for is a highly sensitive test for field diagnosis, not inferior in specificity to PCR,but giving results quickly.

Such characteristics are essential for controlling a coronavirus pandemic or the timely isolation of horses.

Main changes
While working and consulting with the experts, we had to change the design of our project.

Express test for rapid field diagnosis of EHV by CRISPR/dcas9 coupled to beta-lactamase applied to paper

Improved laboratory method for diagnosing EHV-1 and EHV-4 by CRISPR/dCas13 coupled to beta-lactamase and trans-splicing proteins in a plate
How we adapted our system to the values of the project
Our goal in choosing a method is new in synthetic biology and the development of express diagnostics.
Therefore, we tried to adhere to this principle at every stage of the project design
After reading many articles, we got on the phone with Elizaveta Leschiner. During several ZOOM conferences, we learned what was outdated and new in the field of CRISPR-Cas. Finally, we got hooked on the cleaved reporter protein method and began to study it in more detail.
Elizaveta Leshchiner
PhD, Harvard
At the same time, another part of our team was looking for applications of nanoparticles in synthetic biology. Therefore, we contacted the research group of the nanobiotechnology laboratory of MIPT. Here we learned about the successes of nanoparticle diagnostics, parsed their laboratory methods [4], compared them with CRISPR-Cas in the context of synthetic biology. As a result, we found out that the primary goal of all these methods is to increase specificity. Therefore, we chose the method that our team members understand better - CRISPR-Cas methods, as more molecular biologists are among us.

Also, their lab is specialised in creating paper tests. After several discussions, we refused to use them because no one in our team had enough experience, and it was a separate complicated stage. We decided to choose a tablet with diagnostic system solutions.
Maxim Nikitin, head of laboratory nanobiotechnology, MIPT
PhD in Physical and Mathematical Sciences
We discussed different variants of CRISPR-Cas proteins, advised in addition to the originality of the method to think about its feasibility in the laboratories of Russia and cheapness - we started actively looking for laboratories, which published articles on Cas proteins of interest.
Timofei Zatsepin, Skoltech, Moscow
We consulted on the design of our project, the variants of CRISPR-Cas proteins (we compared 9 and 12, 13), how best to select linkers for the reporter protein, which reporter proteins are best to use. The emphasis in the discussion was placed on end-user demands (speed, mobility of the test), diversity of pathogenic herpesviruses (Сas protein must detect the virus of interest).

For greater sensitivity, he recommends combining this also with PCR (try LAMP).

What we did: There are no articles on the viral load of RNA in the saliva of horses with EHV. After talking to an expert on herpesvirus, we decided to be less ambitious in our idea and add a LAMP stage between collection and colorimetric observation of the cleavage of the medium by beta-lactamase. Some articles worked with a minimal load for Cas13. To achieve this, it takes a long time to select the correct gRNAs and conduct statistical testing.

Dmitry Karpov, MSU
PhD, Engelhardt Institute of Molecular Biology, Moscow
We came to the Kurchatov Institute lab with the idea of dCas9 coupled to beta-lactamase halves that detect double-stranded herpesvirus DNA. In discussion, we found that their lab was developing a similar method but with dcas13 with beta-galactosidase. We compared our methods. We decided that it would be easier and cheaper to detect an actively replicating virus by its RNA - so we took their idea with dcas13, despite that it was easier and cheaper to use beta-lactamase. So, we combined both of our methods and started the scientific work in the wet lab.We also added trans-splicing proteins to our assembly to increase diagnostic accuracy.

Maxim Patrushev
PhD, Head of the Kurchatov Genome Center

About choosing viruses for detecting
When a girl on our team reported that her horse had encountered EHV and the virus is severe to the equine world, we immediately began researching and consulting. The Human Practise page details which communities we actively consulted to realize the relevance of creating an equine herpes virus test and to see if it was a significant problem.
We had the following ideas

Diagnose all five herpes viruses by finding a common sequence
Diagnose EHV-1 and EHV-4 as the most dangerous
A system to distinguish EHV-1 SNPs to detect neuro and non-neuro strains
We discussed these questions with experts
Dmitry Karpov:
We discussed all three variants, selected targeting sites (see our bioinformatics work). We found out that the data on neuropathogenicity differ from SNPs. Therefore, the ideal option, both from the point of view of bioinformatics and the relevance of detection, was the second option.

We also discussed it with Dr.Lutz Goehring,Wright-Markey Professor of Equine Infectious Disease. He agreed with our conclusion.

Dr.Lutz Goehring: It is worth starting to diagnose with one. Equine herpesvirus can be confused with other diseases: influenza, arthritis, streptococcus. It is pretty challenging to make a test for one of them, depending on the viral load and the stage of the virus's life cycle. Your idea of doing precisely the RNA test is quite nontrivial and curious.
Test system for equine disease
Our final choice of test system was based on interaction with end-users
Our goals

Easy to use
— CRISPR-Cas system is a new specific method. However, PCR and antibody tests are considered recognized in diagnostics. So, what are the prospects of CRISPR-Cas in this area?
— Official position: A document on the regulation of cases of herpes virus states that tests made only by PCR or antibodies are recognized.

What we think:
Our test will allow doctors to decide rapidly, take measures to prevent outbreaks, and prescribe treatment.
  1. It can be made faster than PCR or antibodies.
  2. That it is pretty specific, and the system with two Cas proteins protects against false-positive results, which increases the specificity.
We understand that CRISPR-Cas tests are not generally accepted, but this is a question of the future.

Our goal is not to entirely abolish the use of PCR tests. Our approach is to reduce the cost and friction of doing a test, which will enable horse owners to perform precautionary tests before symptoms arise at a low cost. When a test is positive, the result is verified by a PCR test and the horse isolated. The rapid test will act as a complementary faster approach to screen the horse population.
What do you think about the CRISPR-Cas 13 system with beta-lactamase and inteins?

Dr.Anna V. Goncharenko, head of the group of microbial genomes editing, The Research Centre of Biotechnology RAS
The idea of using dсas13 with two gRNA guides will minimize the effects of non-targeted RNA guide landings and increase the specificity of the test. Furthermore, reporter beta-lactamase will allow directly registering the binding of guide RNAs to the matrix, which speeds up the diagnosis and does not require additional equipment. In addition, the sensitivity of the detection system will be improved by using intein to assemble parts of the reporter beta-lactamase.

Resources and communities
Our attention was drawn to this problem by a team member, who personally encountered rhinopneumonia of horses.

Naturally, this problem affected her greatly, and we couldn't help but start exploring this area.

We contacted Federation Equestre Internationale (FEI)
They informed us that they were also concerned that the case in Valencia could happen again. We received positive feedback from FEI on developing a mobile test for equine herpesvirus.

For us, the opinion of the organization that directly coped with the largest outbreak of EHV-1 is authoritative, and we consider this a direct confirmation of the relevance of the test. Check this pdf for more information

The Moscow Veterinary Association
We tried to investigate how things are with the diagnosis of rhinopneumonia in Russia.
The state budgetary institution of the city of Moscow, "The Moscow Veterinary Association", performs laboratory-diagnostic and veterinary-sanitary measures to local and eliminate animal diseases.
We found out that they do not conduct diagnostic tests for EHV-1 and EHV-4.
Reports on rhinopneumonia from the Federal Service for Veterinary and Phytosanitary Surveillance
We analyzed the reports on rhinopneumonia from the Rosselkhoznadzor. Their reports show that rhinopneumonia is occured not so often, but the impact is usually essential.

What we think: State authorities collect statistics. Every case of herpes virus disease should be registered at the local veterinary office. We suppose that this is not happening.
The difference between these statistics and communicating with real people is very high.
We have interviewed
Maxima Vet
(veterinary clinic in Moscow)
They have supported us
We noticed that there is a problem in collecting statistics on cases of the herpes virus in Russia. Do you think horse owners hide EHV cases?
We think rhinopneumonia happens. Most horses are vaccinated, but still, sporadic outbreaks are present (diseases with respiratory symptoms and fever). Unfortunately, due to the lack of a quick test to diagnose this disease, it is impossible to determine what it was - herpes virus or something else.

What is the prevalence of the herpes virus in Russia and the world?
The episodicity of outbreaks is on average every 2-3 years. However, sometimes there are more massive outbreaks that affect European countries, we periodically hear about this from foreign colleagues.
What is the peculiarity and danger of the herpes virus in comparison with other viruses?
After contracting a herpes virus infection, many horses retain the respiratory tract's hyperreactivity, which can subsequently turn into chronic obstructive pulmonary disease (COPD).
We know that the existing standards for virus diagnostics are PCR and antibody tests. What do you think about the accuracy of these methods, and how often do you use them for diagnostics?
In practice with horses, paired serums are most often used in Russia, so the diagnosis of the disease takes a month, which has no practical significance. Horses mostly recover after 5-7 days.
Which test would be of interest to the end-user? A fast mobile test, a new laboratory diagnostic method for detecting certain strains?
We would be interested in a fast mobile test and a new laboratory method for detecting certain strains.
What do you think about the vaccine and existing treatments?
There is no specific therapy at the moment. However, available vaccines work under the condition of collective immunity.
The disease is rare, but it is essential to diagnose it firmly in advance so that it does not lead to outbreaks. It is outbreaks that are dangerous.
Lutz Goehring
Head of Equine Medicine and Reproduction at Ludwig-Maximilian University in Munich, Germany.
We interviewed horse owners and veterinarians personally.

All of them confirmed the relevance of the problem and the complexity of existing diagnostic methods.
Herpesvirus is not the most severe problem for horse health. Most often, horses suffer from injuries, helminthiasis. But yes, the herpes virus is common and dangerous. But my horses often go to the races and never catch the herpes virus.
The stable owner (11 horses)
Yes, there is a problem with the herpes virus. We do not know what to do often. The respiratory form is difficult to distinguish from similar diseases, so we are late with the correct treatment. For herpesvirus, the course of the disease is very characteristic without symptoms. Later, the clinical picture develops quickly and is no longer diagnosed, just to save the horse. This is especially true for the nervous form. If the owner reports fevers and that the horse has laid down, it is a herpes virus, and there are 2-3 days left to save the horse. This is precisely the time that diagnostics take, so there is no point in doing this, especially for antibodies.

Antibodies do not show the actual condition of the horse, and their value may change so that we do not understand that the horse is infected. Mucosal PCR tests are always more reliable. But in Moscow, they can only be done at one institute. Therefore, your test, which allows you to do this on the spot and without special equipment, would help you not delay the correct treatment and determine long before the situation becomes irreparable.

Veterinarian of a large clinic in the Moscow region
(private veterinarians also confirmed this information)
According to regulatory documents, each case of rhinopneumonia should be reported to local authorities. For the herpes virus to be entered in the register, the disease must be registered at the local veterinary office, and a referral for diagnosis is received from there. There are cases of concealment and non-registration, which is probably why the official statistics are so low.
Experienced horse owner
There are outbreaks. In the fall and winter of 2017, 2019, and 2020, they were strong. In 2021, also expect closer to November. They are available in and around the Moscow region.
The private doctor from the Moscow region
We dream that the herpes virus can be detected faster in the laboratory. Now we do this mainly for symptoms. The abortive form and the nervous form are clinically distinguishable immediately, but already when sick foals are born, or abortions occur if the nervous one-the horse lies down and starts to get feverish. In this condition, you need to help and treat immediately; time is precious. The selection of medications depends on the correct diagnosis of treatment.

The respiratory form is more complicated. It is easy to confuse it with other viruses that cause flu, arthritis, or just food allergies, and also miss outbreaks.

The general opinion of horse owners
About animals
Picture. Protecting animals by law used for draught and recreation
The map shows the level of animal protection by law used for draught and recreation.
It can be seen that the D level is the highest in the world, which is surprising because the most significant number of people come into contact with these animals [5].

From this, we conclude that the country's legislation is also relatively little interested in animals.

Only in recent years, concerned about the environmental situation, more and more people are raising the issue of caring for animals. As a result, attitudes towards animals have changed.
Our conclusion:
Indeed, there is a problem of insufficient collection of information about ongoing outbreaks. Therefore, we are sure that the situation with the herpes virus in Russia is underestimated.

We agreed that the test should be mobile and precise.
Despite the closed nature of the community, we still managed to get information about outbreaks in Russia and the world, the main problems, and requests.
We are incredibly grateful to everyone who was not afraid to cooperate with us.
We are deeply involved in the community of horse lovers, share their pain, and are eager to create a test and follow-up treatment.

  1. EHV-1 timeline
  2. Field-deployable viral diagnostics using CRISPR-Cas13 doi: 10.1126/science.aas8836.
  3. COVID-19 one year later: a retrospect of CRISPR-Cas system in combating COVID-19 doi:10.7150/ijbs.60655
  4. Nanoparticle Beacons: Supersensitive Smart Materials with On/Off-Switchable Affinity to Biomedical Targets