Biosafety Level 3 Laboratory Research Articles

Inactivation Validation of Ebola, Marburg, and Lassa Viruses in AVL and Ethanol-Treated Viral Cultures.

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log10 TCID50 as the limit of detection.

Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Study on Airflow Distribution and Energy Conservation in a BSL-4 Laboratory Involved in Aerosol Infection Risk

In recent years, COVID-19 has occurred frequently in the world. The biosafety level 4 (BSL-4) laboratory personnel are at risk of being infected by bioaerosols. The high demand for fresh air makes the energy consumption of the laboratory far greater than that of ordinary buildings. This study aimed to propose a suitable air distribution design and reduce the energy consumption of the BSL-4 laboratory, which is beneficial for creating a healthy and green built environment. Firstly, the diffusion characteristics of aerosols, infection risk under different air distributions, and ventilation parameters were analyzed in depth. Secondly, from the perspective of negative pressure control, the influence of parameters such as air tightness and air changes on the airflow in multizone was discussed. The results indicated that the aerosol concentration under the same conditions of the upper supply-upper exhaust is 1.4 times higher than that of the upper supply-bottom exhaust after the airflow stabilizes. The impact of eddy currents on pollutant diffusion is weakened when the air supply velocity reduced from 1m/s to 0.8m/s. Finally, the energy-saving operation strategy was proposed with the optimized ventilation parameters. The results showed that this operation scheme reduced the energy consumption of the laboratory by 15-30%.

A Mouse Model of MHV-1 Virus Infection for Study of Acute and Long COVID Infection.

COVID-19, caused by SARS-CoV-2, has had a significant global impact. While vaccines and treatments have reduced severe cases and deaths, the long-term effects are not yet well understood. Current models used for research, such as non-human primates and transgenic mice, are expensive and require scarce Biosafety Level-3 (BSL-3) laboratories, thereby limiting their practicality. However, the mouse hepatitis virus 1 (MHV-1) mouse model offers a promising alternative. This surrogate model can be investigated in more widely available Biosafety Level-2 (BSL-2) laboratories. Furthermore, mice are affordable and easy to handle, and utilizing MHV-1 as a surrogate for SARS-CoV-2 eliminates the need for costly transgenic mice. Importantly, the MHV-1 model successfully recapitulates COVID-19-related clinical symptoms, weight loss, multiorgan pathological changes and failure in acute stages, irreversible neurological complications, and other long-term organ dysfunction post-infection, which are similar to available human data post-COVID-19. To assist researchers in establishing and using the MHV-1 mouse model, this protocol offers comprehensive guidance encompassing procedures for animal preparation, induction of viral infection, clinical observation, pathological changes, and tissue analysis for mechanistic studies, thereby yielding valuable insights into disease mechanisms and progression. By adopting the MHV-1 model and the provided protocols, researchers can effectively circumvent financial constraints and the limited availability of BSL-3 laboratories, thus facilitating a more accessible and cost-effective approach to investigating the underlying mechanisms of SARS-CoV-2 pathophysiology and exploring potential therapeutic interventions. © 2023 Wiley Periodicals LLC. Basic Protocol: Induction of mouse hepatitis virus 1 (MHV-1) infection in A/J mice Support Protocol 1: Histological evaluation Support Protocol 2: Liver enzyme measurement Support Protocol 3: Western blot analysis of aquaporin expression Support Protocol 4: mRNA measurement Support Protocol 5: Immunohistochemistry/immunofluorescence Support Protocol 6: Tissue water measurement.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

Development of a Novel Positive Pressure Protective Suit for a Biosafety Level 4 Laboratory in Japan.

Biosafety level 4 (BSL-4) laboratories are necessary to study microorganisms that are highly pathogenic to humans and have no prevention or therapeutic measures. Currently, most BSL-4 facilities have suit-type laboratories to conduct experiments on highly pathogenic microorganisms. In 2021, the first Japanese suit-type BSL-4 laboratory was constructed at Nagasaki University. Positive pressure protection suit (PPPS) is a primary barrier that protects and isolates laboratory workers from pathogens and the laboratory environment. Here, we developed a novel PPPS originally designed to be used in the Nagasaki BSL-4 laboratory. We modified several parts of a domestic chemical protective suit, including its front face shield, cuff, and air supply hose, for safe handling of microbiological agents. The improved suit, PS-790BSL4-AL, showed resistance to several chemicals, including quaternary ammonium disinfectant, and did not show any permeation against blood and phages. To validate the suit's integrity, we also established an airtight test that eliminated individual differences for quantitative testing. In conclusion, our developed suit performs sufficiently as a primary barrier and allows for the safe handling of pathogens in our new BSL-4 laboratory.

Study on bioaerosol diffusion and deposition in a mobile BSL-4 laboratory based on air age analysis

Biosafety issues have aroused global concern, especially after the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron strain of corona virus disease 2019 (COVID-19) caused incalculable human and property losses. Laboratory-acquired infections (LAIs) caused by improper operations or accidents are frequently reported. Research is urgently needed for a mobile biosafety level-4 (BSL-4) laboratory with a high risk for exposure. Deposition characteristics and the spatial distribution of bioaerosols under two typical cases were studied in this paper. Based on the age of air and simulation of airflow pattern, a detailed analysis of infection risk and the distribution of bioaerosols was conducted. The deposition characteristics of particles on different surfaces were analyzed based on particle tracking technology. The results showed that the removal rate of bioaerosols was lower in the space area of the laboratory from 1.6 m above the ground. The distribution of high-risk areas is affected by the coupling of equipment layout and pollution sources, mainly located downstream of the main airflow in the laboratory, and the particle concentration was eight times that of the low-risk areas. More than half of bioaerosol particles are deposited on laboratory equipment and walls. The number of particles deposited on the wall was the largest, accounting for 25.02% of the total. The unit area deposition ratio of the experimental table was the highest, which was 6.14 %/m2. The main deposition area of each surface was determined, which could be of guiding significance to the determination of the key disinfection location of the mobile BSL-4 laboratory.

Propagation of SARS-CoV-2 in a Closed Cell Culture Device: Potential GMP Compatible Production Platform for Live-Attenuated Vaccine Candidates under BSL-3 Conditions?

Live-attenuated SARS-CoV-2 vaccines present themselves as a promising approach for the induction of broad mucosal immunity. However, for initial safety assessment in clinical trials, virus production requires conditions meeting Good Manufacturing Practice (GMP) standards while maintaining biosafety level 3 (BSL-3) requirements. Since facilities providing the necessary complex ventilation systems to meet both requirements are rare, we here describe a possibility to reproducibly propagate SARS-CoV-2 in the automated, closed cell culture device CliniMACS Prodigy® in a common BSL-3 laboratory. In this proof-of-concept study, we observed an approximately 300-fold amplification of SARS-CoV-2 under serum-free conditions with high lot-to-lot consistency in the infectious titers obtained. With the possibility to increase production capacity to up to 3000 doses per run, this study outlines a potential fast-track approach for the production of live-attenuated vaccine candidates based on highly pathogenic viruses under GMP-like conditions that may contribute to pandemic preparedness.

Old Enemy with a New Face: Re-emerging Monkeypox Disease – An Update

Human monkeypox (MPX), a multi-country re-emerging disease, is rapidly spreading around the world. The etiological agent of this disease, Monkeypox virus (MPXV), is a DNA virus classified into three genetic types (West Africa, Congo Basin clade, and one new clade-3). Atypical or unusual symptoms as well as asymptomatic infection of MPXV has also been reported. Transmission among humans is possible by droplets, contact, sexual intercourse, and fomites. Secondary transmission of this disease has been reported to occur in less than 10% of cases where it was found 35%–88% of smallpox. Mother-to-fetus transmission by vertical route is also possible for this disease. Modern equipment, biosafety level-3 laboratory facilities, and trained expert persons are needed to diagnose this disease. Previous data support that ~85% clinical protection is provided by smallpox vaccines for monkeypox, although initially non-human primates models were used for various experiments, and also side-effects of this vaccine have been notably mentioned in various studies. Limited research findings of JYNNEOS vaccine has supported the comparatively lower prevalence of MPX cases with vaccination. Few drugs, including cidofovir, tecovirimat, brincidofovir, and vaccinia immune globulin intravenous are preferable against this disease, although clinical trial data is limited and FDA-approval is also pending. This review-based study presents an overall scenario of Monkeypox disease (MPXD) based on previously published studies. Recommended clinical treatment and vaccination, appropriate infection prevention and control strategies, adopting one health approach, and quick identification of hotspots using a wastewater-based surveillance system need to be followed to check the further spread of MPX outbreaks.

A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus.

African Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming. In this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log10HAD50/mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA. The Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.

Intramuscular [18F]F-FDG Administration for Successful PET Imaging of Golden Hamsters in a Maximum Containment Laboratory Setting.

Positron emission tomography (PET) is becoming an important tool for the investigation of emerging infectious diseases in animal models. Usually, PET imaging is performed after intravenous (IV) radiotracer administration. However, IV injections are difficult to perform in some small animals, such as golden hamsters. This challenge is particularly evident in longitudinal imaging studies, and even more so in maximum containment settings used to study high-consequence pathogens. We propose the use of intramuscular (IM) administration of 2-deoxy-2[18F]fluoro-D-glucose ([18F]F-FDG) for PET imaging of hamsters in a biosafety level 4 (BSL-4) laboratory setting. After [18F]F-FDG administration via IM or IV (through surgically implanted vascular access ports), eight hamsters underwent static or dynamic PET scans. Time-activity curves (TACs) and standardized uptake values (SUVs) in major regions of interest (ROIs) were used to compare the two injection routes. Immediately after injection, TACs differed between the two routes. At 60 min post-injection, [18F]F-FDG activity for both routes reached a plateau in most ROIs except the brain, with higher accumulation in the liver, lungs, brain, and nasal cavities observed in the IM group. IM delivery of [18F]F-FDG is an easy, safe, and reliable alternative for longitudinal PET imaging of hamsters in a BSL-4 laboratory setting.

Molecular diagnosis of neglected infectious agents of heep and attle abortions: the prevalences of Coxiella burnetii, Francisella tularensis and Chlamydophila abortus at a glance

Bacterial abortive agents such as Brucella spp., Salmonella spp., Campylobacter spp., Listeria spp. cause serious infections that lead to significant economic losses in cattle and sheep breeding. These bacteria can be easily cultured under in vitro laboratory conditions. Abortions caused by intracellular bacteria such as Coxiella burnetii (C. burnetii), Chlamydophila abortus (C. abortus) and Francisella tularensis (F. tularensis) are less prevalent in abortive cases and the diagnosis of these bacteria, many of which need to cell culture for cultivation and biosafety level-3 laboratory facilities for safe working, can be made by PCR. In this study, it was aimed to determine the molecular prevalence of C. burnetii, C. abortus and F. tularensis agents, which were neglected during the diagnosis of abortions, in cattle and sheep collected from different regions of Türkiye. A total of 395 clinical materials were analyzed via agent-specific commercial Real-Time PCR. As a result, the molecular prevalence of F. tularensis, C. burnetii and C. abortus was determined as 14%, 2.9% and 2.28%, respectively. F. tularensis in cattle clinical samples and C. burnetii in sheep clinical samples were not found. This is the first report that F. tularensis was found as an agent in an abortive material in Türkiye. Although it differs in terms of prevalence, it has been determined that these microorganisms, which are neglected in routine diagnosis, can be spread with aborted materials, especially vaginal discharge, which may pose a risk of transmission.

Mitigation of the biosafety risks of SARS-CoV-2 at BSL-3 laboratories

Aim: The aim of this study is to reduce the biosafety risks of laboratory exhaust air due to SARS-CoV-2 at a biosafety level-3 laboratory used for vaccine development against COVID-19 pandemic. Materials and Methods: In this study, the infrastructure and the organisation of the containment laboratory, which aimed to be used to struggle with pandemic, was used for risk assessment. Assessment of the laboratory exhaust air procedure as a component of the heating-ventilation-air conditioning system, which is significant for high-level biosafety laboratories, was conducted through a risk assessment matrix.

Maintaining differential pressure gradients does not increase safety inside modern BSL-4 laboratories.

This article discusses a previously unrecognized contradiction in the design of biosafety level-4 (BSL-4) suit laboratories, also known as maximum or high containment laboratories. For decades, it is suggested that both directional airflow and pressure differentials are essential safety measures to prevent the release of pathogens into the environment and to avoid cross-contamination between laboratory rooms. Despite the absence of an existing evidence-based risk analyses demonstrating increased safety by directional airflow and pressure differentials in BSL-4 laboratories, they were anchored in various national regulations. Currently, the construction and operation of BSL-4 laboratories are subject to rigorous quality and technical requirements including airtight containment. Over time, BSL-4 laboratories evolved to enormously complex technical infrastructures. With the aim to counterbalance this development towards technical simplification while still maintaining maximum safety, we provide a detailed risk analysis by calculating pathogen mitigation in maximum contamination scenarios. The results presented and discussed herein, indicate that both directional airflow or a differential pressure gradient in airtight rooms within a secondary BSL-4 containment do not increase biosafety, and are not necessary. Likewise, reduction of pressure zones from the outside into the secondary containment may also provide sufficient environmental protection. We encourage laboratory design professionals to consider technical simplification and policymakers to adapt corresponding legislation and regulations surrounding directional airflow and pressure differentials for technically airtight BSL-4 laboratories.

BRET-Based Self-Cleaving Biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery.

ABSTRACTThe 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments, and more inhibitors are under development. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors should be evaluated in Biosafety Level 3 (BSL-3) laboratories. Here, we design DNA-coded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After overexpression of the biosensors in human embryonic kidney (HEK) 293T cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their previously reported anti-SARS-CoV-2 activities. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery.IMPORTANCE The virus proteases 3CLpro are validated drug targets for developing antivirals to treat coronavirus diseases, such as COVID-19. However, the development of 3CLpro inhibitors relies heavily on BSL-3 laboratories. Here, we report a series of BRET-based self-cleaving biosensors that can be used to set up cell-based assays to evaluate the cell permeability and cellular activity of SARS-CoV-2 3CLpro inhibitors in BSL-2 laboratories. The cell-based assay is suitable for high-throughput screening for 3CLpro inhibitors because of the simplicity and good reproducibility of our biosensors. The design strategy can also be used to design biosensors for other viral proteases for which the activation processes involve the self-cleavage of polyproteins.

Novaferon Effectively Inhibits Ancestral SARS-CoV-2 and Omicron Variant inVitro, 2022.

IntroductionTo identify Novaferon (Nova), a novel recombinant protein of interferon (IFN)-α, antiviral activity against ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Omicron variant in vitro. MethodsVero cells were infected with SARS-CoV-2 and Omicron variant in a biosafety level-3 laboratory. And viral replications were accessed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).ResultsResults demonstrated that Nova has effective inhibition against ancestral SARS-CoV-2 and Omicron variant in vitro. DiscussionThe in vivo effects of Nova need to be further tested in animal models. And large-scale randomized double-blind clinical trials are needed to reveal its potentially clinical application.

Bactericidal efficacy of a low concentration of vaporized hydrogen peroxide with validation in a BSL-3 laboratory

Highly infective pathogens are cultured and studied in biosafety laboratories. It is critical to disinfect these laboratories thoroughly in order to prevent laboratory infection. A whole-room, non-contact, reduced corrosion disinfection strategy using hydrogen peroxide was proposed and evaluated. To evaluate the bactericidal efficacy of 8% and 10% vaporized hydrogen peroxide (VHP) in a laboratory setting, with spores and bacteria used as bioindicators. Spores of Bacillus atrophaeus and Bacillus stearothermophilus, along with Escherichia coli, Staphylococcus aureus and Staphylococcus albus bacteria were placed in pre-selected locations in a sealed laboratory, and an OXY-PHARM NOCOSPRAY2 VHP generator was applied. Spore killing efficacy was evaluated qualitatively, bactericidal efficacy was analysed quantitatively, and the mean log10 reduction was determined. Finally, the optimized disinfection strategy was verified in a biosafety level 3 (BSL-3) laboratory. Significant reductions in microbial load were obtained for each of the selected spores and bacteria when exposed to VHP 8% and 10% for 2-3h. S.aureus was found to be more resistant than E.coli and S.albus. Tests with VHP 8% and exposure for >3h showed a 100% kill rate for B.atrophaeus on surfaces and equipment in the BSL-3 laboratory. The VHP generator has good diffusivity and low corrosiveness, and is a time-saving method for removal of disinfectant residue. This study provides reference for the precise disinfection of air and the surfaces of objects in biosafety laboratories under various conditions.

Development of a positive pressure protective suit for BSL-4 laboratory

Biosafety Level 4 (BSL-4) laboratories are required for research on microorganisms that are highly pathogenic to humans and for which there are no prevention or treatment methods. Currently, the majority of BSL-4 laboratories in more than 60 around the world are suit-type laboratories using positive pressure protective suits. In 2021, the first suit-type BSL-4 laboratory in Japan was constructed at Nagasaki University. Positive pressure protective suits are important as primary barriers to protect workers from pathogens, but the selection process has been largely unexplored. Here, I describe the selection process for the positive pressure protective suits to be used at the BSL-4 laboratory of Nagasaki University, and introduce a novel positive pressure protective suit (PS-790BSL4-AL), which was originally designed and produced in Japan.

Developing a Biosafety Level 4 Laboratory user training program

In recent years, numerous emerging and reemerging infectious diseases have occurred worldwide and have seriously threatened our society. As a countermeasure against the pathogens responsible for serious diseases (classified as class 4 pathogens), we are preparing for full operation of the first suit-type biosafety level 4 (BSL-4) facility available for basic and applied research at Nagasaki University. For the safe operation of these facilities, experienced and qualified personnel with appropriate skills and knowledge of biorisk management must be certified. Developing an appropriate training system is a prerequisite for ensuring the safety of users involved in research in a BSL-4 laboratory. Here, we introduce an overview of the content of the training program that we are currently establishing for the BSL-4 facility at Nagasaki University. We are designing this program to follow national and international guidelines and regulations in part by referring to experiences and materials derived from multiple BSL-4 facilities in other countries. The established training program system, including the formulation processes, will serve as a reference and will provide practical materials for other research organizations to develop their own high-containment laboratory training programs.

Biorisk Management for SARS-CoV-2 Research in a Biosafety Level-3 Core Facility.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents hazards to researchers and other laboratory personnel in research settings where the live virus is stored and handled. The Biosafety Level-3 (BSL-3) Core Facility (CF) at Yong Loo Lin School of Medicine in National University of Singapore (NUS Medicine) has implemented a biorisk management (BRM) system to ensure that biorisk to employees, the public, or the environment are consistently minimized to an acceptable level while working with SARS-CoV-2. This chapter summarizes how a BRM system can be implemented in academic institutions based on international standards in the context of existing local legislations/regulations and institutional policies/guidelines to minimize the risk of laboratory-acquired infections and deliberate misuse of the newly emerged virus, SARS-CoV-2 in BSL-3 laboratories. The BRM programs prioritize performing risk assessments prior to implementation of work processes and reassessing the risk portfolio of the facilities from time to time, determining root causes and prevention of recurrences. Focusing on awareness-raising and educating the laboratory users in biosafety and biosecurity, and identifying opportunities for improvement are the other key factors for a sustainable and successful BRM system in the NUS Medicine BSL-3 CF.