Our project aims to develop a novel antiviral defense mechanism that prevents the amplification of the virus within a host organism by actively inducing programmed cell death in influenza virus-infected cells.

For this purpose, we needed to conduct experiments involving the infection of animal cell cultures with the virus to provide a Proof-of-Concept. However, as viral infection experiments are outside the iGEM White List, we are required to devise a secure plan and submit a Check-In Form.

We faced considerable difficulty determining what to consider when drafting the experimental plan to ensure the safety of the infection experiments.

This document outlines the essential elements that an iGEM team must incorporate into a safety plan when conducting viral infection experiments in animal cell culture. By referencing this document, future iGEM teams will be able to draft secure experimental plans with minimal effort and gain the necessary approval from the Safety Committee for their activities.

A significant number of animal cell culture lines are designated as Risk Group 2 under the iGEM Safety guidelines[1]. Consequently, the submission of a Check-In Form and the acquisition of authorization from the Safety Committee are mandatory.

While animal cell cultures may appear safe at first glance, leading to the potential oversight of this requirement, it is imperative that teams correctly submit the Check-In Form prior to their use.

When submitting the Check-In Form for cell cultures, it is crucial to clearly articulate their specific experimental application or purpose within your project.

As an illustrative example, we will now detail the experimental plan and safety protocols that our team submitted in our Check-In Form.

Our Experiments Plan

This experiment serves as a safe, virus-free proof of concept by externally introducing double-stranded RNA (dsRNA) into cells to mimic viral stimulation. This approach enables us to simulate key aspects of viral infection without using actual viruses, thereby establishing a foundational demonstration of our system. To achieve this, the animal cells used in the experiment have been engineered to express the designed fusion protein through three distinct delivery methods.

1. Transient Transfection: Introducing plasmids encoding the fusion protein.
2. Stable Cell Lines: Creating cell lines that constitutively express the fusion protein.
3. Direct Protein Introduction: Purifying the fusion protein from E. coli and delivering it into the cells, reproducing the methods from the reference publication.

Our system is composed of designed fusion proteins that link a dsRNA sensor to an apoptosis inducer.

1. dsRNA Sensors: RIG-I or PKR
2. Apoptosis Inducers: APAF1 or iCaspase9

All of these proteins are derived from Homo sapiens, Gallus gallus, or Anas platyrhynchos. Importantly, none of them are toxic to humans.

To validate the dsRNA-dependent function, we observe for apoptosis after introducing dsRNA (made by in vitro transcription) to cells that have received the fusion protein. The dsRNA is prepared according to the publication[2], using the process approved in our Check-In Form.

All components are non-toxic to humans. The safety of this approach is supported by A prior publication confirming the function of a similar PKR-APAF1 fusion[3]. Use of pre-approved parts, including the iGEM safe part HIV TAT (BBa_K1202006) and a modified RIG-I with its interferon-inducing domain removed.

Safety Measures

All experiments involving animal cells (and not viruses) are directly supervised by our team's instructor, Dr. Shinnosuke Honda. All experiments are conducted in Dr. Shinnosuke Honda's laboratory, which is part of the Graduate School of Agriculture at Kyoto University and is highly proficient in genetically manipulating animal cells.

Furthermore, only members who have been instructed by Dr. Shinnosuke Honda and have learned how to handle animal cells will be directly involved in the experiments.

Experimental activities involving viruses are not on the iGEM White List[4]. Consequently, we submitted a Check-In Form and proceeded with the work only after receiving authorization from the Safety Committee.

The key points that iGEM teams should consciously address and document in the Check-In Form during this process are as follows:

1. Point 1: Is the viral strain to be used suitable for handling in a laboratory environment of BSL-2 or lower?
2. Point 2: Is it possible for the Biosafety Level (BSL) to be raised to BSL-2+ / BSL-3, even if the viral strain used is originally classified as BSL-2 or lower? If so, under what circumstances might this possibility occur?
3. Point 3: When using multiple viral strains, what measures will be implemented to prevent cross-host infection?
4. Point 4: Detailed information on the laboratory facility where the infection experiments will be conducted and the training status of the team members.

Points 1 and 2 are often complex due to varying regional and institutional regulations, making independent investigation difficult. Therefore, you should first consult with the researchers affiliated with the laboratory where the infection experiments will take place.

Point 3 highlights crucial precautions to prevent the emergence of mutant viruses during the experiment.

Regarding Point 4, you must detail the information about the infection facility and provide a thorough description of the training plan for team members who will perform the viral infection experiments.

As an example, we have summarized the information that our team submitted in our Check-In Form below.

Evidence that the Viruses Used are Classified as BSL-2 or Lower

The two types of influenza viruses used were the Human Influenza Virus and the Low Pathogenic Avian Influenza Virus.

The specific viral strains are listed below:

Human Influenza Virus

• A/WSN/33 (H1N1)
• A/California/04/2009 (H1N1pdm)
• A/Victoria/361/2011 (H3N2)

Loq PAthogenic Avian Influenza Virus

• A/duck/Hokkaido/8/1980 (H3N8)
• A/swan/Hokkaido/481102/2017 (H4N6)
• A/duck/Hokkaido/95/2014 (H8N4)

All these strains are commonly used in laboratory settings, are known to have extremely low pathogenicity (virulence), and pose a minimal risk of causing infection or harm to the experimenter.

Therefore, the experiments can be performed safely by any team member who has completed BSL-2 handling training, even without professional knowledge concerning viruses.

Is it Possible for the BSL to be Raised to BSL-2+/BSL-3, Even When the Used Viral Strain is BSL-2 or Lower? If so, Under What Circumstances?

In Japan, the handling Biosafety Level (BSL) for pathogens is determined by specific regulations for each pathogen. For influenza viruses, classification is based on serotypes; all serotypes except H5 and H7 are permitted for handling at BSL-2[5]. Therefore, all influenza virus strains included in this application can be safely handled in a BSL-2 laboratory. Furthermore, within the scope of our proposed experiments, Japanese laws[6] indicate that the legal BSL of the virus will not increase during experimental procedures, allowing us to consistently conduct experiments in a BSL-2 facility.

Generally, a virus's BSL is elevated when its pathogenicity increases through experimentation.According to Japanese law[6], specifically regarding livestock, viruses that meet the following criteria are treated as BSL-3. If a BSL-2 virus were to meet these pathogenicity criteria during an experiment, it could be reclassified as BSL-3 after an appropriate review process.

This applies only to viruses that meet any of the following criteria (1 to 3), excluding equine influenza viruses and novel influenza viruses:

1. An Intravenous Pathogenicity Index (IVPI) exceeding 1.2 in 6-week-old chickens.
2. A mortality rate of 75% or more in chickens aged 4 to 8 weeks upon intravenous inoculation.
3. In addition to criteria 1 and 2, a serotype of H5 or H7, possessing multiple basic amino acids at the cleavage site of the hemagglutinin molecule, and an amino acid sequence presumed to be similar to a pathogen confirmed under criterion 1 or 2."

However, in the current experiments, we anticipate no increase in BSL from BSL-2 to BSL-2+ or BSL-3 for four reasons:

• We will conduct the experiments within a scope that does not induce enhanced virulence through cross-species infection.
• We will not perform experiments that artificially induce mutations in the virus.
• We will not conduct animal infection experiments or large-scale culturing.
• Influenza viruses other than H5 or H7 serotypes do not show increased pathogenicity in cell-level experiments.

Thus, we conclude that the risk of inducing significant enhancement of virulence in the viruses is zero for this specific experimental protocol.

Measures to Prevent Cross-Host Infection

In including multiple viral strains in the experimental plan, we will conduct experiments by paying attention to the following matters.

First, in our experiments, we will not co-infect two different viral strains into one cell. To prevent unintended viruses from infecting the cells during the infection experiments, we will thoroughly clean the clean bench with each experiment and conduct the experiments while carefully isolating the samples. We will never handle two types of viruses simultaneously.

Furthermore, the work of independently handling multiple viral strains is routinely conducted in Professor Noda's laboratory, which is our host laboratory, and we plan to proceed with the experiments cautiously in accordance with their strict guidance. These experiments have been conducted with approval from the Kyoto University Recombinant DNA Experiment Safety Committee, and adhere to the Kyoto University Regulations Regarding Safety Control of Recombinant DNA Experiments, etc. and the Enforcement Rules for the Kyoto University Regulations Regarding Safety Control of Recombinant DNA Experiments, etc. [7][8].

Details of the Laboratory Facility and Personnel Training Status

The host laboratory where all viral infection experiments will be conducted is the laboratory of Dr. Takeshi Noda at the Institute for Frontier Life and Medical Sciences, Kyoto University:

This laboratory is certified by the university-wide Safety Committee of Kyoto University as possessing facilities that meet the standards for conducting BSL-2 experiments. In addition, it also has operational BSL-3 facilities, as it sometimes handles highly pathogenic viruses.

Under the guidance of this laboratory, team members completed BSL-2 experimental training before the start of the experiments.

The total number of members engaged in the infection experiments was two: one member familiar with DNA work and animal cell handling, and one member familiar with DNA work. Each of them will undergo training for BSL-2 level experiments at the Noda Laboratory before engaging in the main experiments of the project. The training was conducted on August 18, 19, and 20, 2025. The training includes instruction on the handling of Noda Laboratory's equipment and aseptic techniques using the clean bench. Furthermore, the Noda Laboratory independently conducts a viral handling safety lecture for Kyoto University students, and the members performing the experiments will also attend this lecture. Moreover, during the experiments, a supervisor who is accustomed to BSL-2 level experiments at the Noda Laboratory will constantly monitor the team to prevent accidents. In addition, hazardous experimental procedures that require significant operational proficiency will be entrusted entirely to this supervisor, ensuring that the team members do not perform dangerous manipulations.