Infection detecting sysytem

Over view

In order to distinguish the four serotypes of dengue virus, we focused on neutralizing antibodies. We used particles called "Single Round Infectious Particles (SRIPs)" to test for the presence of neutralizing antibodies in sera. They cannot replicate even if infected. There are two things we create in this system. The first is the SRIPs corresponding to the four dengue virus serotypes. The second is a cell to detect SRIPs infection. First, SRIP detecting cells need to behave differently when infected with SRIPs than when uninfected with SRIPs. We wanted to show the difference in behavior by using different fluorescent proteins expressed in the cells. We employed the Cre-LoxP system as a system to switch between the two: green fluorescent protein, EGFP, is expressed when the Cre-LoxP system is not activated, and red fluorescent protein, mCherry, is expressed after the Cre-LoxP system is activated. However, Cre is somewhat too long to be placed on the genome of single round infectious particles (SRIPs), as inferred from the 2018 iGEM TokyoTech project results. Therefore, we decided to solve this problem by using Split-Cre [2], which is a two-part split of Cre: the C-terminal region of Cre recombinase enzyme (hereafter referred to as “C-Cre”) is incorporated into the genome of SRIP, and the N-terminal region of Cre recombinase enzyme (hereafter referred to as “N-Cre”) is produced in the SRIP detecting cells. The cells producing the particles were also treated with a new method to detect the infection. The particle-producing cells were HEK293T cell and C6/36 cell derived from the Streptococcus pyogenes, which are known to frequently produce viruses of the Flaviviridae family, while Vero cell and C6/36 cell were used for the SRIP detecting cells.
The particles produced here "do not contain genes for structural proteins" and "have C-Cre". Cells for infection detection are characterized as "expressing N-Cre and EGFP in the absence of SRIP infection" and "when the Cre-LoxP system functions, N-Cre and EGFP are removed and mCherry is expressed.

Fig.1: SRIP is not infectious if there are neutralizing antibodies in the serum. If there are no neutralizing antibodies, SRIP is infectious.

Using this system, we test for the presence of neutralizing antibodies to the four serotypes of the Dengue Virus. If, as shown in the figure, only type 2 SRIPs have ever been infected, and if there are neutralizing antibodies in the serum that act only against type 2, type 2 SRIPs are prevented from infecting SRIP detecting cells and remain expressing GFP, but types 1, 3, and 4 SRIPs infect SRIP detecting cells, which in turn express mCherry. SRIP detecting cells express mCherry.

Fig.2: Which serotypes of SRIP are not infectious?

Background

Existing method advantages and disadvantages

Several mechanisms already exist to diagnose each serotype.

Table 1: Comparison of detection methods

The advantages and disadvantages of existing methods are explained in the following links. Integrated Human Practice

Dr. Suzuki's method

Dr. Suzuki of the National Institute of Infectious Diseases (NIID) has cooperated with us in this project. Dr. Suzuki's system is as follows. First, the genomes of the Flaviviridae family are composed of sequences encoding Capsid, Membrane, and Envelope, which constitute the viral particle itself, and non-structural protein sequences, which encode proteins necessary for replication and other processes. These sequences were divided into Capsid, prME (precursor membrane and envelope) and non-structural protein, and the genomic sequences of Nano-Luciferase and autolytic peptides were attached to the non-structural protein. By co-transfection of these plasmids into HEK293T cells, HEK293T cells produce single-round infectious particles (SRIPs) that do not encode any structural proteins. If these SRIPs infect Vero cell, Nano-Luciferase is expressed from the Nano-Luciferase in the particle and luciferin is emitted; Nano-Luciferase is expressed, but as we have said before, the sequence encoding the structural protein is not enclosed. No particles are produced from this Vero cell.

Fig.3: split and insert(Dr. Suzuki's Method) Fig.4: Overview of Dr. Suzuki's method

2018project

Based on Dr. Suzuki's method, iGEM TokyoTech has conducted a project in 2018 Team:TokyoTech 2018 wiki. Instead of Nano-Luciferase, they inserted different fluorescent protein genes into each of the four dengue virus serotypes: EGFP in DENV1, DsRed in DENV2, ZsYellow in DENV3, and AmCyan in DENV4. However, in this project, only EGFP and AmCyan showed fluorescence after SRIPs infection. After discussion with Dr. Suzuki and PI Prof. Tagawa, we concluded that this was due to the fact that the fluorescent protein gene inserted in place of Nano-Luciferase was too long and SRIPs did not form normally, resulting in the production of many empty SRIPs in which no genome was inserted.

Fig.5: by TokyoTech-2018 wiki

Our new method

System Description

We planned this project based on the above results (iGEM2018 TokyoTech). Based on the consideration that the sequence length of the fluorescent protein on the particles may have been too long, we decided to use an inducer with a sequence length shorter than that of the fluorescent protein and to prepare SRIP detecting cells corresponding to the inducer as well as the particles. The Cre-LoxP system and Split-Cre were chosen for this purpose. The Cre-LoxP system was used as the mechanism for the "SRIP detecting cell corresponding to the inducer," and Split-Cre was used as the "inducer with a sequence length shorter than that of the fluorescent protein. In addition, a nuclear localization signal (NLS) is attached to each Split-Cre to facilitate the assembly of Split-Cre that are translated at different locations. This allows the Cre-LoxP system to recombine portions of the genome within SRIP detecting cells when SRIPs infect them, resulting in changes in cell behavior. Specifically, we designed the system to change the expressed fluorescent protein from EGFP to mCherry. Fig.6: Overhead view of the system

By using the "four SRIPs corresponding to dengue virus serotypes" and the "SRIP detecting cell," it is possible to determine whether neutralizing antibodies are present in the specimen.

Why do we even use C6/36 cell?

In this study, we used cells derived from the tiger mosquito( Aedes albopictus ), called C6/36. The reason for using C6/36 cell is that Flaviviridae viruses, which are transmitted by mosquitoes, are replicated and produced more frequently in the cells of mosquitoes, the intermediate host, than in the cells of humans, the terminal host, and therefore, the inducer may function more rapidly and SRIPs may be produced more frequently. We therefore decided to use the HEK293T cells and C6/36 cells as the host cells for the study. Therefore, we used human codon-optimized synthetic DNA for HEK293T cell and Vero cell and mosquito codon-optimized synthetic DNA for C6/36 cell. In the future, we would like to discuss the significance of codon optimization, and would like to generate cells transformed with plasmids constructed from human codon-optimized synthetic DNA for C6/36 cell.

References
[1] Yamanaka A, Matsuda M, Okabayashi T, Pitaksajjakul P, Ramasoota P, Saito K, Fukasawa M, Hanada K, Matsuura T, Muramatsu M, Shioda T, Suzuki R. Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012. mSphere. 2021 Aug 25;6(4):e0033921. doi: 10.1128/mSphere.00339-21. Epub 2021 Jul 14. PMID: 34259560; PMCID: PMC8386448
[2] Rajaee M, Ow DW. A new location to split Cre recombinase for protein fragment complementation. Plant Biotechnol J. 2017 Nov;15(11):1420-1428. doi: 10.1111/pbi.12726. Epub 2017 Apr 20. PMID: 28317293; PMCID: PMC5633763.
[3] Matsuda M, Yamanaka A, Yato K, Yoshii K, Watashi K, Aizaki H, Konishi E, Takasaki T, Kato T, Muramatsu M, Wakita T, Suzuki R. High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon. Sci Rep. 2018 Nov 9;8(1):16624. doi: 10.1038/s41598-018-34865-y. PMID: 30413742; PMCID: PMC6226426.