Identifying the problem

We began our research on the problem of dengue fever in April of this year. It is often said that it is difficult to prevent the spread of the dengue virus because there is currently no vaccine. Even a cursory Google search reveals that there is not enough data on dengue virus infection, and with four different serotypes of dengue virus, it is difficult to predict which serotype will be the next epidemic, making it difficult to effectively distribute vaccines. We are working on a project to solve these problems.
We set out to solve these problems, but since dengue fever is not a common infectious disease in Japan, we needed to know how dengue fever is actually detected in the areas where it is prevalent. First, we talked to a doctor who is familiar with the areas where dengue is prevalent.

Overall Overview

First interview with Dr. Kameoka

Point：We gained a deeper understanding of the local reality of dengue fever detection. We also gained tips on how to improve the systems we use.

Photo 1: Meeting with Dr. Kameoka

First, regarding the local diagnosis, the internist at the affiliated local hospital told us that diagnosis and treatment are done according to the guidelines. In addition, they said that definitive diagnosis, such as measurement of NS-1 (nonstructural protein of dengue virus) or neutralizing antibodies in serum, is almost never performed. The main reason why these definitive diagnoses are rarely made is that antibody tests are expensive, costing 1,500 yen per test. The "serotype diagnostic kit" similar to the one we are planning to produce is not widely used for diagnostic purposes. We were also told that the accuracy of diagnostic kits like the one mentioned here is not very high.

From these factors, we decided to create a "kit for research purposes, not for diagnostic purposes," and to create a serodiagnostic kit that is more sensitive than "antigen test kits as they currently exist.

Next, we asked him about the usefulness of serotype diagnostic kits. Dr. Kameoka's experience through his research to date is that the severity rate increases when the prevalent serotypes are switched, so there may be a need for serotype testing kits themselves. In addition, he said that PCR is basically used for serotyping in the field, but since PCR requires a clinical bench and other equipment, it is difficult to conduct serotyping in areas where it is difficult to introduce such equipment. In addition, there is a need for serotyping in order to predict epidemics, but due to equipment and financial problems, symptomatic treatment is the only way to go.

From these facts, we learned that "there is a definite need for our diagnostic kits" and that our diagnostic kits "need to be made in such a way that they do not require large machinery.

We also asked for your feedback on our project at this point.

If Single-round infectious particle (SRIP) is used, the accuracy of SRIPs will be reduced if the antibody is carried by particles of membrane and outer membrane proteins without nucleocapsid, since dengue tends to produce only membrane and outer membrane proteins without nucleocapsid.
Detection of fluorescence is a bottleneck, so other devices may be possible if a certain degree of fluorescence is observed.
The antibody test is better after recovery of symptoms, but the antigen test is better during treatment.

This advice was useful in our experiments, and the perspective that the use of the kit was different compared to antigen test kits that can detect NS-1, IgM, and IgG was something that did not exist for us before, and was very useful in considering how the project would actually be implemented in the society.

First interview with Dr. Suzuki

Point:We received some hints regarding why the 2018 project did not work. This had a huge impact on the root of our project.

We spoke with Dr. Suzuki, the author of the original paper on this system based on the failures of 2018.In the 2018 project, we were unable to observe the expected fluorescence when the cells for infection were infected with SRIP.Regarding the reason for this, Dr. Suzuki pointed out the length of the genes to be placed in the SRIP.Dr. Suzuki's system included a gene encoding Nano-Luciferase in the SRIP, whereas the 2018 system included a gene encoding a fluorescent protein.Dr. Suzuki said that the gene encoding the fluorescent protein is larger than the gene encoding the Nano-Luciferase, so it may not have been successfully incorporated into SRIP.

These factors led us to think about how to reduce the size of the genes in the SRIP.

Interview with iGEM Tokyo Tech 2018 Team

Point: After receiving tips from Dr. Suzuki on how to improve the system, and after discussions with the iGEM Tokyo Tech 2018 team, we decided to use Split-Cre.

Photo 2: Meeting with iGEM Tokyo Tech 2018 team

After Dr. Suzuki pointed out that the length of genes to be included in the SRIP may have been too long for the 2018 project, iGEM Tokyo Tech 2018 members and iGEM Tokyo Tech 2022 members had a discussion on how to shorten the length of genes to be included in the SRIP The opinions of our seniors who have graduated from the iGEM team and are now working in laboratories had a great impact on our project. Among them, Mr. Miyamoto's suggestion to use Split-Cre, in which the Cre enzyme is divided into an N-terminus and a C-terminus, became the basis of our project. By using Split-Cre, we can shorten the length of the gene to be inserted into SRIP.
After this discussion, we decided to use Split-Cre to shorten the length of genes to be included in SRIP.

Interview with Dr. Nukuzuma

Point: We learned that the use of C6/36 cells, which can be cultured at room temperature, could be very useful for the social implementation of this project.

As we gradually worked out the project details, we faced two problems. The first is that an incubator is required to culture the cells used in the detection kit. This would not allow us to achieve our goal of creating a detection kit that does not require large machinery. Second, animal cells may not be able to successfully produce SRIP, and we have raised the possibility that the gene length in the SRIP was too long as a reason why the 2018 project did not go well, but there were other opinions that SRIP production in animal cells may not be successful. I discussed this with Dr. Nukuzuma. When I discussed this with Dr. Nukuzuma, he suggested that since the dengue virus originally multiplies in mosquito cells, it might be more efficient to use mosquito cells to produce SRIP. Dr. Nukatzuma suggested using C6/36 mosquito cells, which can be cultured at room temperature, and using C6/36 cells and HEPES buffer, which allows pH control independent of CO2 concentration, it may be possible to culture them in the field. This may make it possible to cultivate the cells in the field. Also, using mosquito cells to produce SRIP may allow for more efficient production.

Based on these facts, we decided to conduct experiments using C6/36 cells, which can be cultured without the need for large machinery and may (or may not) be capable of producing SRIP at high efficiency.

The second interview with Dr. Suzuki

Points:We learned that our detection method definitely has higher throughput than existing detection methods and that it is difficult to use SRIP-based detection methods in the field.

Photo 3: Meeting with Dr. Suzuki

We asked Dr. Suzuki, who referred us to his paper for our detection method, several questions about this system.

Question1:Is our detection method high-throughput compared to existing detection methods "because the switch to fluorescence-based assays allows us to measure in 96 wells"?

-I think that is certainly one of the major factors.In addition to this, however, another factor is the short time required for detection.After the virus and antibody suspension is poured into the cell monolayer, it can take as long as 5~7 days for the plaque to form.In addition to this, it is necessary to use a dish with a somewhat large body surface area to observe the plaque.This makes it difficult to employ a testing method that allows testing of many specimens at once instead of a small bottom area such as 96 wells, in addition to the fact that it takes a lot of time for plaque to form.This makes the SRIP-based detection method high-throughput compared to existing detection methods.In addition, although there are machines that automatically count the plaque, in many cases the human operator counts the plaque by eye. This makes the human labor to perform the detection very large, which is another drawback of the plaque assay.

Question2:For example, RT-PCR method allows real-time measurement by fluorescence without electrophoresis of the final nucleic acid, but is it possible to perform real-time assay by fluorescence like RT-PCR in this system?

ーWe do not perform real-time measurements in our experiments, but some researchers are currently doing so.You may want to shorten the time to measurement by measuring in real time, and we have found that in this system, significant results can be obtained in 24 hours or even 12 hours.Even if a very small amount of SRIP is infected compared to the plaque assay, this detection kit fluoresces, so real-time measurements may be possible.

Question3:This detection method using SRIP we would like to eventually do in the field, do you think it is possible?

ーI think it will be difficult.When we created SRIP, we envisioned that it could lower BSL, not that it could be used in the field.For example, the PRNT method requires a BSL-3 level laboratory, but it may be difficult for some laboratories to prepare a BSL-3 level laboratory due to the cost of installation.It may be possible to perform the assay in BSL-1 or BSL-2 laboratories in these locations if the assay is performed using SRIP.In addition, since serum separation would be required, centrifugation would be necessary, but this would also be difficult to perform in the field, which is a concern.

Interview with Shimadzu Corporation

Points:In creating a device that detects fluorescence, we spoke with manufacturers who actually create various devices and received feedback.

Photo 4:Meeting with Shimadzu Corporation (see the Hardware-Discussion page for details).

Symposium at QWS

We collaborated with iGEM Waseda team and SHIBUYA QWS to organize a symposium on synthetic biology on August 12 this year. At this event, we invited Professors Daisuke Kiga and Toru Asahi of Waseda University, Nobuhiro Hayashi of Tokyo Institute of Technology, and Shishin Kawamoto of Hokkaido University, and invited high school students, undergraduate students, graduate students, and adults to discuss "What is synthetic biology," "What does it do, what are the risks," "How is it We held an online introduction and discussion on "What is synthetic biology," "What does it do, what are the risks," and "How can we implement it in society?” Photo 5: Joint symposium with Waseda University

In this symposium, we hope to promote synthetic biology to a wide range of people, to ask how our detection method might look to those who do not normally detect infectious diseases, and to encourage participants to think about the problem we are aiming to solve: how to assay for dengue virus in a low-cost, simple, and sensitive way. We hoped for flexible advice from people outside of iGEM by introducing our project and asking them to think about the challenge we are aiming to solve: how to assay dengue virus with lower cost, simplicity, and sensitivity.

Figure 1: Our Aim

Some of the results of the survey are presented below.

The wide range of ages and professions represented in the survey indicates that we were successful in communicating synthetic biology to people of various demographics without dividing them.

Figure 2:Age of participants

Figure 3:Occupation of participants

In addition, the following is a partial excerpt from a questionnaire about the benefits and underlying dangers of synthetic biology .

Figure 4:Results of the Synthetic Biology Questionnaire

We expected that those who were uneasy about synthetic biology would believe that synthetic biology was dangerous and that strong regulation was necessary, but in fact this was not generally the case, so we learned that the barriers to promoting synthetic biology are not simple.

(see Education for details).

Conclusion

The following is a summary of what we have learned through this activities.

The idea was to create SRIP with Cre enzyme fragments inside.
The perspective of using mosquito cells to increase the efficiency of SRIP production while allowing it to be used in the field.
Know-how in actually creating the hardware
Public Perspectives on Synthetic Biology By obtaining various opinions, we believe we were able to improve our project and bring it closer to social implementation.

Creation of a dengue virus detection kit

Main Points.
Understand current problems.
Explore detection methods.
Comparison with current detection methods.
Prospects for the future.

Main points

Issues to be addressed

More user-friendly and sensitive detection kits need to be produced to operationalize the vaccine.
Currently, large and expensive equipment is often required to detect dengue virus serotypes.
Tests to detect dengue virus serotypes cost more than $10 and and can be often expensive for people in areas where dengue is endemic.

Our Problem Solution

Development of detection methods that do not require expensive, large machinery or BSL-2 level equipment.
Development of detection methods that are inexpensive per test and do not require difficult operations.

Figure 1. Our solution

Understand current problems.

After hearing from Dr. Kameoka of Kobe University, who is familiar with areas where dengue virus is prevalent, we learned that local people would not accept a detection method that is too expensive for a single test, that it is necessary to develop a test that does not require a large machine, and that it is necessary to develop a test kit for research use that is more sensitive than existing products. In order to create a model for vaccine application, Dr. Kameoka said it is necessary to solve these problems and develop a test kit with better sensitivity than the current one.

Figure 2. Our Approach

Explore detection methods.

Our goal has consistently been to "reduce the number of people who will suffer from dengue fever in the future" rather than to "save those who are currently suffering from the dengue virus. To this end, we looked into the current research dengue fever diagnostic kits available. Then we found that most of them use antibodies to detect antigens (NS-1) and antibodies (IgG and IgM). This method is relatively inexpensive compared to PCR and other methods, and does not require large-scale machinery. Currently, simple detection kits employ an antibody-based detection method called immunochromatography. We felt that this detection kit was less sensitive than PCR and insufficient for our goal of accumulating data for each serotype.
Figure 3. Immunoassay is not sufficient Therefore, we spoke with Dr. Yoichi Tagawa, Associate Professor at the Tokyo Institute of Technology, who is an expert in virology. According to Dr. Tagawa, sensitivity can be improved by using cultured cells instead of proteins (antibodies). Unlike immunochromatography, which only embeds proteins, cultured cells self-renew, so even if the amount of the biomarker is small, the cells will proliferate and emit a lot of fluorescence as time passes. The cells should be able to emit a lot of fluorescence as time passes, even if the amount of the biomarker is small.
Figure 4. Fluorescence assay is better We decided to use living cells in our detection kit. The first candidate was E. coli. However, E. coli can spread when tested in the field. We considered using kill switches and other spread prevention measures, but we decided on a more fundamental and reliable method. That is the use of cultured animal cells in the detection kit. However, if cultured cells are used in the detection kit, they will not be able to self-propagate even if they are released into the natural world, and thus will not spread.
Figure 5. Diffusion does not occur Next we started thinking about what substances we would detect with our detection kits. After referring to existing simple test kits and PCR and PRNT methods, we have narrowed down the candidates to two substances to be detected. They are NS-1 and neutralizing antibodies. NS-1 is a nonstructural protein produced by dengue virus and detected in patient sera during infection. Neutralizing antibodies are proteins produced in the body of patients infected with dengue virus that have the ability to recognize and bind to antigens. The NS-1 is primarily used to test whether a patient is currently infected with dengue virus. This makes it impossible to identify a history of infection and is incompatible with our kit. For these reasons, we decided to create a kit to detect neutralizing antibodies. Considering that the kit will be used to determine the history of dengue infection in a large number of people, neutralizing antibodies that remain in the body after infection are better suited for detecting infection history than NS-1, which is no longer detectable in the blood immediately after infection.
As described below, the PCR and PRNT methods are commonly used to detect serotypes, but these instruments are large and may require a BSL-2 level laboratory. Therefore, our design of a kit to detect neutralizing antibodies will be of great benefit to countries that lack the equipment and funding to conduct large-scale studies for future dengue virus research, as well as to insurance organizations participating in UNITEDengue, the dengue virus community.

Fig.6:Putting in shorter sequences Based on these ideas, we set about designing a specific detector. Our senior iGEM Tokyo Tech 2018 team was aiming to create the same dengue virus serotyping kit as we did. The results, however, were hardly a complete success. The plan was to use fluorescence to determine infection with SRIP, which is a fake viral particle, but several serotypes could not be observed to fluoresce well.
So we spoke with Dr. Suzuki of the National Institute of Infectious Diseases, who is an expert in virology and our mentor. Dr. Suzuki suggested that the reason why it did not work was that we put a sequence that was too long within the SRIP. The referenced paper used a gene encoding nanoluciferase, whereas the 2018 project had a gene encoding a fluorescent protein in the SRIP.
With these things in mind, we discussed with iGEM Tokyo Tech 2018 members how to shorten the sequences to be introduced in SRIP and we came up with Split-Cre. Split-Cre makes it possible to create a test kit similar to the one originally planned while shortening the sequence to be introduced into the SRIP. However, one problem arose here. The animal cells used in the test kit require a carbon dioxide incubator in order to be cultured. This would not achieve our goal of developing a detection kit that does not require large machinery. So, we spoke with Dr. Nukuzuma. Dr. Nukuzuma suggested that we develop a detection kit using mosquito cells called C6/36 cells. Since C6/36 cells can be cultured at room temperature, the use of HEPES buffer, which is pH-regulated independent of CO2 concentration, may allow them to be cultured in the field. He also said that dengue virus increases well in mosquito cells, which may increase the efficiency of SRIP production.

Figure 7: Using C6/36 cells Through these circumstances, we decided to perform a high-throughput assay of neutralizing antibodies using Split-Cre and SRIP. This detection kit is very suitable for our goal of field testing for dengue virus serotypes, and is inexpensive, simple, and highly sensitive compared to conventional detection kits, and does not require large machinery. We have also begun development of software and hardware that will enable even novice users to easily detect fluorescence in cells, which has been our goal from the beginning. The new detection kit produces fluorescence that enables quantitative analysis of the presence of viruses. Fluorescence is captured by hardware. Software has also been developed to perform these detections.

Comparison with current detection methods.

Existing methods for dengue virus detection include

RT-PCR
Immunoassay
Enzyme-Linked Immunosorbent Assay（ELISA）
PRNT etc. How is our detection method superior to these methods?

RT-PCR

RT-PCR is a highly sensitive and specific assay to detect viruses by amplifying the nucleic acids of viruses present in the specimen. Although this assay is very common, one of the characteristics of flaviviruses is that they do not appear in blood very often. Therefore, the possibility of infection cannot be ruled out even if the serum collected during RT-PCR is negative. In addition, most cases of dengue fever are asymptomatic, so even if the test is performed after the patient becomes aware of symptoms, the amount of virus in the sample may already be low, and infection may not be successfully determined. Therefore, there is a need for a specific and sensitive serological method for the detection of flaviviruses. Existing serological tests are described below.

Figure 8: How PCR works

Immunoassays and ELISAs

These detection methods are prone to false positives because of cross-reactivity between flaviviruses or between dengue viruses of different serotypes. This makes immunoassays and ELISAs difficult to use in epidemiological studies of flaviviruses. For these reasons, the plaque assay has been considered the gold standard for flavivirus detection.

Figure 9: Sandwich ELISA

How PRNT works

In the PRNT method, the serum or other sample to be tested is diluted and mixed with a viral suspension, and the antibody reacts with the virus. This is then poured into a monolayer of cells, and the degree of plaque formation is observed. This method is very complicated and involves many steps, and it takes 4 to 7 days for plaques to form. Also, the number of samples that can be examined at one time is small. However, cross-reactivity is also detected in the PRNT method, so tests for multiple serotypes of dengue virus must be performed to specifically detect dengue virus. However, as mentioned above, this method is not high-throughput. To solve this problem, we have developed a high-throughput neutralizing antibody detection method using SRIP.

Figure 10: How PRNT works

Summary

It is our belief that our detection method is super compatible with the PRNT method. It retains the good points of the plaque assay while allowing for a higher throughput assay. The advantages of our detection method are as follows

Lower BSL and possible because we do not use real virus
Fluorescence-based assay enables detection in 96-well plates for high throughput.
Shorter detection time compared to the PRNT method
Some viruses are difficult to obtain for the PRNT method, but with our detection method, there is no need to order viruses from the supplier.

Table 1: Comparison of detection methods

Future Prospects

One issue we are facing is "the grade of laboratory required to use SRIP ''. The PRNT method, currently the most commonly used neutralization test, requires a BSL-3 level laboratory. SRIP, however, can certainly be performed in a lower laboratory. We considered using this detection kit in the field, but Dr. Suzuki of the National Institute of Infectious Diseases suggested that it might be difficult to use the SRIP outdoors.

The wet system in our detection kit is very promising. This detection kit can be adapted to many viruses once we have the sequence of the structural protein of the virus. For example, the PRNT assay has been used in epidemiological studies of novel coronaviruses, and we believe that our detection kit will be useful for vaccine development and clinical use in regions and laboratories where the PRNT assay is difficult to implement.

Integrated Human Practices

Human Practice