Introduction

Through the recent pandemic of COVID-19, we realized the importance of preventing infectious diseases. While COVID-19 has been prevalent globally, we realized that tropical infectious diseases also affect many mid-latitude regions including Japan. In Tokyo,Japan, people were confused by an outbreak of dengue fever, one of the tropical diseases, in 2014. According to the Centers for Disease Control and Prevention, 400 million people are infected with dengue virus every year and 40,000 people die.A paper presenting the quantified future risk of dengue fever in Japan under climate change states that under the RCP8.5 scenario, dengue fever could be endemic in Japan by 2100 (Katsuma Hayashi et al.,2022) We ran the project, "Establishment of dengue virus serotype prediction and detection systems," to prevent dengue virus transmission in 2018. This project is safe and can be applied to other viruses, but only 60% of the project was completed in 2018. Therefore, we hope to improve and complete this project this year for the prevention of dengue transmission.
Dengue fever is caused by the dengue virus (DENV) in the flavivirus family. It is transmitted to humans by female mosquitoes, Aedes aegypti. There are four serotypes, and infections with multiple serotypes can often cause severe dengue. (Fig.1)

Dengue Virus four serotypes
Figure 1.Mlutiple infection: Four serotypes of dengue virus with different structures of protein fragments (top) and process of complications after the cross-infection to other serotypes. These clinical perspectives come from interviews with physicians living in dengue-endemic areas and with virologists.(For more information, please follow Integrated Human Practice section)

Problem statement

It is crucial to predict serotypes for prevention because it contributes to the research of treatments or supplements for vaccines, but the low local diagnosis rate is a problem. The existing diagnostic tools cannot achieve all the diagnostic requirements: sensitivity, specificity, and applicability for flavivirus (Table 1). As a result, we can hardly plan efficient vaccine distribution due to the lack of serotype data.

Table 1.Status quo of existing diagnostic tools: RT-PCR (Reverse transcription PCR), immunoassay, ELISA (enzyme-linked immunosorbent assay) and PRNT (plaque reduction neutralization test).

Solution

To tackle the limitations of the diagnostic methods, we aimed to create a highly-sensitive method for identifying the infection serotype of the dengue virus from a blood sample. We also created prediction models of the serotype’s ratio which can be combined with the results from the wet lab experiments (Fig. 2).
Figure 2.Wet Lab-Dry Lab:Scheme of predicting the ratio of dengue virus’s serotypes based on the interaction between prediction models and serotype detection tools

Overview of project

The final goal of our project is to predict the prevalent serotypes of dengue virus and contribute to the development and distribution of vaccines and drugs. To achieve this goal, we need data on the number of people infected with each serotype and software that can predict the prevalent serotypes. To accumulate data on the number of people infected with each serotype, it is necessary to develop an easy-to-use dengue virus serotype infection detection kit. Our dengue virus serotype infection detection kit can be monitored by fluorescence. However, measuring fluorescence generally requires expensive equipment, which may hinder the widespread use of our detection kit. Therefore, we decided to also develop a fluorescent measurement device to measure the fluorescence of our detection kit.

We created the following tools (Fig. 3). (1) Dengue Virus serotype detection kit (Wet lab) (2) Fluorescent measurement device (Hardware) (3) Epidemic Serotype Prediction System (Dry lab) Figure 3.Work flow:Our diagnostic scheme of the dengue virus’s four serotypes toward efficient allocation of vaccines. (Created with BioRender.com)

Overview 1: Dengue Virus Serotype Infection Detection Kit

We produced Single-Round Infectious Particles (SRIPs) and SRIP detecting cells.
The SRIPs include each epitope proteins corresponding to the serotype of dengue virus. First, three plasmids encoding the SRIP component were transfected into HEK293T cells or C6/36 cells and SRIPs were collected. Second, we produce SRIP infection detecting cells. SRIP infection detecting cells produce GFP, green fluorescent protein, but SRIP infection detecting cells produce only mCherry, red fluorescent protein. The SRIPs including each epitope proteins corresponding to the serotype of dengue virus(Fig. 4)
Figure 4.Wet Work Flow:System of SRIP and SRIP infection detecting cell.

When we mix this SRIP with the blood serum of an infected person, the infectivity of one type of SRIP is blocked by antibodies of a specific serotype with a history of infectionAs a result, only EGFP is produced. On the other hand, other three types of SRIPs can infect the cells, then mCherry is produced. Therefore, the serotype-positive SRIP-infected cells have the red fluorescence, but negative ones have the green fluorescence. (Fig. 5).
Figure 5-1.Detection Kit system:Mechanism of neutralization assay with single-round infectious particles (SRIP) Figure 5-2.Detection Kit Example:Detecting a specific serotype of dengue virus based on fluorescences.
Wet lab-Design

Overview 2: Fluorescent Measurement Device

The fluorescence measurement device is a machine that can determine whether a fluorescent is present or absent. Our dengue virus serotype serotype infection detection kit needs to detect the fluorescence of green fluorescent protein (GFP) and red fluorescent protein (mCherry). So, our fluorescence measurement device enables users to use the infection detection kit without expensive fluorometers.
This device has three parts. First, it has a physical environment where we can observe fluorescence in every condition. Second, it has an excitation light and color filters so that we can capture fluorescence with a camera. Third, it can analyze the image comparing negative control with it and tell us whether there is fluorescence. To make our device, you do not need something special, you will just need things easily available, such as a camera, plastic board, black paper, LED light and color cellophane. That is why if you want to know whether there is fluorescence, it will be easier and cheaper to use our device than to use other devices for medication or experiments. (Fig. 6).
Figure 6.Scheme of tailor-made fluorescence analyzer
Hardware

Overview 3: Epidemic dengue virus serotype prediction system

The Epidemic dengue virus serotype prediction system can accumulate data on infectious serotypes diagnosed with our infection serotype detection kit and predict the number of people infected with each serotype based on several years of data on the number of people infected with each serotype. This system can be used as a web application by people who are not familiar with programming. Two steps were taken to create this system.

• Step1:Development of the modeling method for predicting epidemic serotypes.( Modeling)

• Step 2: Implement the modeling developed in Step 1 into the web application for predicting epidemic serotypes.(Software)

  Figure 7.Time-series trends in infection cases with different serotypes of dengue virus

The advantages of our project

Infection serotype infection detection kit

• It is more accurate than conventional serotype tests
• It does not require expensive machines or special techniques.
• it does not use BSL-2 facilities. (This is because the kit uses SRIPs, not viruses.)

Flourescent Measurement Device

• It does not require expensive machines to observe fluorescence.

Epidemic dengue virus serotype prediction system

• Even people who are not familiar with programming can use the epidemic dengue virus serotype prediction system.

Reference

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