Experiment

Establishment of dengue virus serotype detection system

Abstract

To identify which serotype of dengue virus your target patient is infected with, many methods have been explored and established. Since our prediction model requires the certain number of consensus time-scale data, it has been urgent to develop a high-throughput and cost-efficient tool for the serotype identification.

With the assistance of Dr. Suzuki at National Institute of Infectious Diseases, our team designed a new detection system with fluorescence protein. Three types of constructs (Genetic feactures: 1. C, 2. prM-E and 3. Fluorescence protein-FMDV2a-NS) should be prepared first. After that, they are introduced into HEK293T cells and pseudo-viruses are produced after a certain interval. They are added to cultured Vero cells to check the infectivity by measuring the intensity of fluorescence emitted from fluorescence protein that works as mono-cistron.

Since each one of the constructs contains either structural or non-structural region, the pseudo-virus just causes single-round infection. In terms of biosafety, our system and products that apply this system can be demonstrated even in the real world.

Background

RT-PCR, ELISA and neutrilization assay - these methods have been widely used to identify infection experience and its serotype. However, RT-PCR is valid only for 0 to 5 days after infection and ELISA and neutralization assay have problem with cost-efficiency.

Figure 1: Change in viral load and antibodies (Source: Guzman, M. G. et al., 2010)

Figure 2: Comparison of dengue detection system with existing ones

Experiment Overview

Our experiment consists of three parts: 1. Construction, 2. Pseudo-virus Production and 3. Pseudo-virus Infection. After three types of constructs are introduced into HEK293T cells, pseudo-viruses are produced. They are added to cultured Vero cells, and you can check the infection after a certain interval.

Figure 3: Experiment Overview (iGEM Tokyo Tech 2018)

1. Construction

For the pseudo-virus production, we prepared structural gene, and non-structural gene with fluorescence protein gene.

About structural gene, capsid (C), membrane (prM) and envelope (E) are necessary for the formation of viral structure. We prepared pCAG-C and pCAG-prM-E for Serotype I to IV.

About non-structural gene with fluorescence protein gene, we prepared EGFP-FMDV2a, DsRed-Express-FMDV2a, ZsYellow-FMDV2a and AmCyan-FMDV2a, and inserted each of them to non-structural genes of dengue virus.

Figure 4: Construct line-up and original genes derived from dengue virus

2. Pseudo-virus Production

We introduced pCAG-C, pCAG-prM-E and FP-FMDV2a (FP: Fluorescence Protein) into HEK293T cells. After cell culture for about 2 days, we collect the supernatant that contains pseudo-viruses produced from HEK293T cells.

Figure 5: Basic flow of transfection and pseudo-virus production

3. Pseudo-virus Infection and Functional Evaluation of Pseudo-virus

After collecting pseudo-viruses from HEK293T cells, we added them to Vero cells. When they can successfully infect the cells, the enclosed fluorescence protein gene is coded and we tried to estimate the amount by measuring the fluorescence intensity. Since we split up the structural region and non-structural region, the pseudo-viruses produced in our system can infect host cells only once. Thus, it will not spread out of the system and will not be harmful.

Figure 6: Basic flow of pseudo-virus infection and how fluorescence protein works

Note: Validated Parts (Silver Medal Criteria)

DENV2 C, EGFP-FMDV2a and DsRed-Express-FMDV2a are essential for pseudo-virus production. As one of the structural genes, DENV2 C plays an important role in virus structural formation. EGFP-FMDV2a and DsRed-Express-FMDV2a are enclosed in pseudo-virus and programmed to code functional fluorescence proteins after infecting the host cells. To get specific sequence and charanterization information, please see the following Parts Pages. Parts Page: DENV2 C Parts Page: EGFP-FMDV2a Parts Page: DsRed-Express-FMDV2a

Figure 7: List of validated parts and the features

Progress of Our Experiment (~Oct. 17th, 2018)

Our team successfully validated that all the necessary constructs are produced by using electrophoresis (please refer to our Parts Pages). After completing the construction, our team shifted to the transfection into HEK293T cells for pseudo-virus production. Using lipofection, the constructs have been already introduced into the cells and the pseudo-viruses will be collected in a few days. After collecting the pseudo-viruses, they will be added to cultured Vero cells. Our team will test the infectivity by measuring the fluorescence intensity at the end. The whole results will be announced in Giant Jamboree, so please do not miss it!

Our Goal

We successfully validated that the pseudo-viruses are produced, and they can infect the host cells. Making the most of our system and lab experience, our team will make a kit to identify which type your target patient was infected with before. As you can see in Fig. 8, if the patient was infected with type II virus before, type II-specific antibodies in blood serum decrease the infectivity of type II pseudo-virus. As a result, fluorescence protein programmed in the pseudo-virus would not be coded so much due to the decrease in the number of infectious viruses. Thus, you can identify the infection experience and its type by measuring or observing the fluorescence intensity for each type/color.

Figure 8: Mechanism of colorimetric type detection system with different fluorescence proteins

Feedback to Modeling

Since our detection system enables us to collect more samling data in many areas quickly and with low cost, our prediction model can be more precise thanks to the increase in the consensus time-scale data of dengue-affected patients and serotype ratio.

Biosafety

As you can see in Fig. 9, on native virus genome, structural genes and non-structural genes are integrated on the same sequence. Thus, the whole genome are replicated and that make viruses possible to replicate themselves in host cells.

However, as you can see our construct design in Fig. 10, structural and non-structural regions are splitted so that the pseudo-virus cannot replicate themselves in host cells because RNA polymerase from non-structural V gene cannot replicate structural genes.

To sum up, our system starts and ends in one place and doesn't harm environment.