Safe viral chassis
Our team chooses vaccinia virus as the chassis for bioengineering. Poxviruses replicate within specialized cellular organelles that are only established post infection, significantly reducing the likelihood of viral DNA integration into the host genome, thereby preventing host gene disruption and oncogenic effects (1).
When generating Click Virus, we replaced codons of three phenylalanines at the N-terminus of A27L with amber suppressor codons (A27L-3Stop). Such modification not only allowed us to incorporate 4-azido-L-phenylalanne to A27L as the click-reaction sites, but also enhanced the safety of click virus in use. VACV has two morphologically distinct infectious virions: intracellular mature virus (IMV), which resides in the cytoplasm, and extracellular enveloped virus (EEV), which is released to the extracellular environment for viral dissemination (2). A27L is important for the formation of EEV but not IMV. Deficiency of A27L leads to a dramatic reduction of EEV and thus impairs viral dissemination (3). When Click Virus is used, the progeny viruses cannot synthesize complete A27L due to the lack of artificial translation system, leading to a drastic reduction in EEV formation, which greatly decreases the infectiousness and pathogenicity of the modified virus. Viruses unable to synthesize complete A27L mostly remain at the IMV stage, unable to spread over long distances, thereby ensuring the safety of the experiment and its applications (Figure 1).
Figure 1. Mechanism of A27L-3Stop enhancing the safety of Click Virus in use.Without the artificial translation system, the progeny virus cannot synthesize complete A27L, leading to a reduction in EEV formation, which decreases viral ability of dissemination.
Biosafety facilities
All virus-related experiments in our project were conducted in a Biosafety Level 2 (BSL-2) laboratory. The BASL-2 laboratory（Figure 2） is divided into a clean area, a semi-contaminated area, and a contaminated area, with anterooms. It also has an independent ventilation and disinfection systems(Figure 3). This setup not only meets experimental requirements but also ensures the health of laboratory personnel.Our team's used biosafety cabinet is of Class II Type B2(Figure 4), which features a high-efficiency filtration system and a negative pressure environment(Figure 5). It uses HEPA filters to filter the air within the cabinet, All exhaust gases are rigorously channeled through High-Efficiency Particulate Air (HEPA) filters to maintain sterility and prevent cross-contamination
Figure 2. BSL-2 Laboratory Preparation Area
Figure 3. Biosafety System
Figure 4. Biosafety Cabinet
Figure5 Pressure Detector
Standard Operating Procedure (SOP)
All laboratory personnel must undergo operational training before conducting experiments(Figure 6、7). When conducting virus experiments, any equipment that comes into contact with the virus must be disinfected with a bleach solution after use(Figure 8).In the process of item conveyance to the laboratory, our team follows the protocol of unidirectional transport and rigorously subjects the materials to high-pressure sterilization to ensure biosecurity and prevent cross-contamination(Figure 9), and strict 15-minute UV sterilization procedures are carried out after the experiments.
Figure 6. Laboratory Application and Usage Procedure Regulations
Figure 7. Laboratory Management and Operation-Related Documents
Figure 8 Beach and Medical Waste Bin
Figure 9. Pass-through Window or Transfer Hatch
Personal protective equipment (PPE)
When conducting virus experiments, every person should wear proper PPE(Figure 10), including mask, gloves, and specific lab coats(Figure 11、12). Experiments are conducted inside a biosafety cabinet.This provides a safe, sterile working environment for laboratory personnel.
Figure 10. Personal Protective Equipment (PPE)
Figure 11. Changing Room
Figure 12. BSL-2 Laboratory Personal Protective Equipment Storage Area
Safety in Use(SIU)
Our click virus does not require genetic modification during use, making it simpler and more convenient to operate, and also safer. The modified click virus is incapable of producing complete progeny viruses, and thus, it will not cause tissue damage due to the uncontrolled proliferation of progeny viruses during use(Figure 13).
Figure 13. Comparison of the Safety of the Original Virus and Our Modified Virus
Biosafety Promotion
Our team has meticulously facilitated comprehensive biosafety education for all members, ensuring adherence to laboratory safety protocols and individual operational safety. Furthermore, we have endeavored to enhance public biosafety awareness by developing a biosafety manual(Figure 14、15), engaging in outreach programs targeting secondary school students(Figure 16), and disseminating the manual through our team's digital platforms. These initiatives aim to empower a broader audience with the knowledge and practices essential for biosafety.
Figure 14. Biosafety Manual Outer Packaging
Figure 15. Biosafety Manual Internal Display Diagram
Figure 16. Comparison of the Safety of the Original Virus and Our Modified VirusFigure 15 Team members give a lecture at Jincheng No.2 Middle School.
Measures we put in place
Firstly, we have completed an exceptionally rigorous laboratory safety training program. To mitigate any risk of leakage or contamination, our vaccinia virus stock is meticulously stored in sealed containers within a dedicated virus storage room. All experiments involving vaccinia viruses are meticulously conducted in an authorized BSL-2 laboratory. The detailed procedures are as the Laboratory Safety Manual.
Our insistence on choosing vaccinia virus as the reason
Firstly, vaccinia virus Tian Tan（AF095689.1）is considered the most secure virus within our laboratory setting, having been continuously cultured over an extended period. And we have developed sophisticated and refined methods for the genetic modification of the vaccinia virus, enabling us to tailor its properties for various applications including reserch, vaccine development, and therapeutic uses. It is important to note that, while our modification techniques are highly advanced and effective, their application is specific to the vaccinia virus. These methods do not readily translate to other viral species, due to the unique genetic and structural characteristics that differentiate vaccinia from other viruses. Furthermore, this virus has found substantial application in various domains, notably in vaccine development and as an oncolytic virus for cancer treatment. Importantly, upon infecting a host, vaccinia virus instigates a disease that is self-limiting, meaning it resolves on its own without the need for medical intervention.
Demo video
We have filmed a video about personal protection in a BSL-2 laboratory.
References
1. Kaynarcalidan O, Moreno Mascaraque S, Drexler I. Vaccinia Virus: From Crude Smallpox Vaccines to Elaborate Viral Vector Vaccine Design. Biomedicines. 2021 Nov 26;9(12):1780. doi: 10.3390/biomedicines9121780.
2. Vanderplasschen A, Hollinshead M, Smith GL. Intracellular and extracellular vaccinia virions enter cells by different mechanisms. J Gen Virol. 1998;79 ( Pt 4):877-87.
3. Vazquez MI, Esteban M. Identification of functional domains in the 14-kilodalton envelope protein (A27L) of vaccinia virus. J Virol. 1999;73(11):9098-109.