High-containment Laboratories Research Articles

Enhancing Biosafety and Biosecurity: Quality Management in High-Containment Laboratories

Quality Management in high-containment laboratories plays a pivotal role in ensuring the safe handling of biological agents and toxins, thereby mitigating potential biorisks. This paper provides a comprehensive exploration of the key aspects of Quality Management Systems (QMS) tailored to the unique challenges of high-containment laboratories. It delves into the significance of QMS in enhancing biosafety and biosecurity measures, safeguarding laboratory personnel, the community, and the environment. The paper also discusses the integration of international standards, risk management strategies, and the role of top management in fostering a culture of safety. Through this examination, it becomes evident that a robust QMS not only ensures compliance but also promotes continual improvement and innovation in high-containment laboratory operations, ultimately advancing the field of biosafety and biosecurity.

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories.

Pathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. SARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. Heat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. By ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

Development of an EBOV MiniG plus system as an advanced tool for anti-Ebola virus drug screening

The incidence of zoonotic diseases, such as coronavirus disease 2019 and Ebola virus disease, is increasing worldwide. However, drug and vaccine development for zoonotic diseases has been hampered because the experiments involving live viruses are limited to high-containment laboratories. The Ebola virus minigenome system enables researchers to study the Ebola virus under BSL-2 conditions. Here, we found that the addition of the nucleocapsid protein of human coronaviruses, such as severe acute respiratory syndrome coronavirus 2, can increase the ratio of green fluorescent protein-positive cells by 1.5–2 folds in the Ebola virus minigenome system. Further analysis showed that the nucleocapsid protein acts as an activator of the Ebola virus minigenome system. Here, we developed an EBOV MiniG Plus system based on the Ebola virus minigenome system by adding the SARS-CoV-2 nucleocapsid protein. By evaluating the antiviral effect of remdesivir and rupintrivir, we demonstrated that compared to that of the traditional Ebola virus minigenome system, significant concentration-dependent activity was observed in the EBOV MiniG Plus system. Taken together, these results demonstrate the utility of adding nucleocapsid protein to the Ebola virus minigenome system to create a powerful platform for screening antiviral drugs against the Ebola virus.

Cyberbiosecurity in high-containment laboratories.

High-containment laboratories (HCLs) conduct critical research on infectious diseases, provide diagnostic services, and produce vaccines for the world's most dangerous pathogens, often called high-consequence pathogens (HCPs). The modernization of HCLs has led to an increasingly cyber-connected laboratory infrastructure. The unique cyberphysical elements of these laboratories and the critical data they generate pose cybersecurity concerns specific to these laboratories. Cyberbiosecurity, the discipline devoted to the study of cybersecurity risks in conjunction with biological risks, is a relatively new field for which few approaches have been developed to identify, assess, and mitigate cyber risks in biological research and diagnostic environments. This study provides a novel approach for cybersecurity risk assessment and identification of risk mitigation measures by applying an asset-impact analysis to the unique environment of HCLs. First, we identified the common cyber and cyberphysical systems in HCLs, summarizing the typical cyber-workflow. We then analyzed the potential adverse outcomes arising from a compromise of these cyber and cyberphysical systems, broadly categorizing potential consequences as relevant to scientific advancement, public health, worker safety, security, and the financial wellbeing of these laboratories. Finally, we discussed potential risk mitigation strategies, leaning heavily on the cybersecurity materials produced by the Center for Internet Security (CIS), including the CIS Controls®, that can serve as a guide for HCL operators to begin the process of implementing risk mitigation measures to reduce their cyberbiorisk and considering the integration of cyber risk management into existing biorisk management practices. This paper provides a discussion to raise awareness among laboratory decision-makers of these critical risks to safety and security within HCLs. Furthermore, this paper can serve as a guide for evaluating cyberbiorisks specific to a laboratory by identifying cyber-connected assets and the impacts associated with a compromise of those assets.

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses.

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.

Marburg virus disease outbreak amid COVID-19 pandemic: an emerging concern in Ghana, West Africa.

Marburg virus disease outbreak amid COVID-19 pandemic: an emerging concern in Ghana, West Africa.

Improving U.S. Biosafety and Biosecurity: Revisiting Recommendations from the Federal Experts Security Advisory Panel and the Fast Track Action Committee on Select Agent Regulations.

In response to a series of biosafety incidents in 2014, the White House directed two high-level expert committees to analyze biosafety and biosecurity in U.S. laboratories and make recommendations for work with select agents and toxins. Overall, they recommended 33 actions to address areas related to national biosafety, including promoting a culture of responsibility, oversight, outreach and education, applied biosafety research, incident reporting, material accountability, inspection processes, regulations and guidelines, and determining the necessary number of high-containment laboratories in the United States. The recommendations were collected and grouped into categories previously defined by the Federal Experts Security Advisory Panel and the Fast Track Action Committee. Open-source materials were examined to determine what actions had been taken to address the recommendations. The actions taken were compared against the reasoning provided in the committee reports to determine if the concerns were sufficiently addressed. In this study, we found that 6 recommendations were not addressed and 11 were insufficiently addressed out of 33 total recommended actions. Further work is needed to strengthen biosafety and biosecurity in U.S. laboratories handling regulated pathogens (biological select agents and toxins [BSAT]). These carefully considered recommendations should now be enacted, including determining if there is sufficient high-containment laboratory space for response to a future pandemic, developing a sustained applied biosafety research program to improve our understanding of how high-containment research should be performed, bioethics training to educate the regulated community on the consequences of unsafe practices in BSAT research, and the creation of a no-fault incident reporting system for biological incidents, which may inform and improve biosafety training. The work presented in this study is significant because previous incidents that occurred in Federal laboratories highlighted shortcomings in the Federal Select Agent Program and the Select Agent Regulations. Progress was made on implementing recommendations to address the shortcomings, but efforts were lost or forgotten over time. The COVID-19 pandemic has provided a brief window of interest in biosafety and biosecurity, and an opportunity to address these shortcomings to increase readiness for future disease emergencies.

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.

High-level biocontainment laboratories: risks and necessity for society

Advancements in the biological sciences have made it possible to manipulate life forms in unprecedented ways. Recognizing the possible dangers connected with this activity, as well as with work involving natural pathogens, countries have promoted the building of High Safety and High Containment Laboratories, classified as Biological Safety Levels 3 and 4. In this article I briefly summarize the major features of these laboratories, exemplify some of the research that they host, highlight the possible dangers, and argue for the opportunity of a reduction of possibly dangerous research, and for more transparency and openness about activities that imply risks not only for those involved, but for human and environmental health as well.

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory

The COVID-19 pandemic has highlighted the critical role that animal models play in elucidating the pathogenesis of emerging diseases and rapidly analyzing potential medical countermeasures. Relevant pathologic outcomes are paramount in evaluating preclinical models and therapeutic outcomes and require careful advance planning. While there are numerous guidelines for attaining high-quality pathology specimens in routine animal studies, preclinical studies using coronaviruses are often conducted under biosafety level-3 (BSL3) conditions, which pose unique challenges and technical limitations. In such settings, rather than foregoing pathologic outcomes because of the inherent constraints of high-containment laboratory protocols, modifications can be made to conventional best practices of specimen collection. Particularly for those unfamiliar with working in a high-containment laboratory, the authors describe the logistics of conducting such work, focusing on animal experiments in BSL3 conditions. To promote scientific rigor and reproducibility and maximize the value of animal use, the authors provide specific points to be considered before, during, and following a high-containment animal study. The authors provide procedural modifications for attaining good quality pathologic assessment of the mouse lung, central nervous system, and blood specimens under high-containment conditions while being conscientious to maximize animal use for other concurrent assays.

Decontamination of High-Containment Laboratories for Demolition: A Risk-Based Approach.

Introduction:Decontamination of redundant laboratories, contained plant space, and support buildings used to handle high consequence animal pathogens (North America BSL-3Ag, UK SAPO4) was required before demolition to mitigate the risk that infectious material was released into the environment.Methods:Given the age of the buildings and their construction, bespoke qualitative risk-based methods were developed by biorisk personnel, in consultation with specialist contractors where applicable. This approach was to give assurance that suitable decontamination was achievable, sometimes through multilayered approaches to disinfection. Time was considered as a contributing factor in decontamination. Different means of achieving decontamination were employed, and waste management was considered as part of the overall process ensuring a “cradle to grave” approach.Summary:This article describes the challenges and solutions, faced by a UK facility licensed to work on high hazard pathogens, including foot-and-mouth disease virus and African swine fever virus. The risk-based approach (and methods described herein) may also be applicable to routine decontamination of younger buildings, that is, during renovations or temporary derestriction.

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods.

Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses.

Strengthening Biorisk Management in Research Laboratories with Security-Sensitive Biological Agents Like SARS-CoV-2.

In this chapter, we discuss potential incidents associated with SARS-CoV-2 experimental work in high containment research laboratories. The risk landscape in high containment laboratories is changing due to the strong innovation drive of the life sciences research. Thus, the WHO has recommended life sciences organizations to incorporate good research practices and ethical principles into a risk-based approach of the biorisk management (BRM). Currently, BRM systems in high containment laboratories are predominantly steered by operational personnel and laboratory professional. It is well known that without having a systematic approach and leadership support from the organization, the BRM system in the high containment laboratory will not be sustainable. Even though the roles of organizations and their leadership in establishing the BRM system are spelt out in many international standards, guidance documents and national legislations, operational aspects of these roles are rarely discussed.It is therefore important for everyone to understand about their roles in organizational processes (communication, decision, and performance evaluation) involved in implementation of BRM related operational activities. In this chapter, discussion is based on operational activities of four main organizational behaviors that are considered to have strengthened BRM systems in high containment laboratories: (1) displaying a visible commitment and support to the BRM system from different levels of management, (2) developing a competent and responsible workforce with BRM technical skills and problem identification/solving skills, (3) integrating learning and improvement principles into the BRM system, and (4) enhancing the continuous motivation of laboratory personnel to avoid complacency. The categorization of these organizational behaviors is based on the International Atomic Energy Agency's principles and guidance for strengthening the safety and security culture in nuclear facilities. Furthermore, we encourage the laboratory management to identify gaps in processes and activities related to those organizational behaviors so that one could rapidly address biosafety and biosecurity vulnerabilities in high containment laboratories.

Introduction of high containment laboratories in abroad

Recently, outbreaks of highly pathogenic viruses, such as those of Ebola and Lassa viruses, have become a global public health issue. Such viruses must be handled in biosafety level 4 (BSL-4) laboratories. Currently, 62 BSL-4 laboratories are in operation, under construction, or planned in 24 counties. In this review, I provide an overview of the current status and characteristics of BSL-4 facilities in abroad and introduce my research on the wild-type Ebola virus at the BSL-4 facility in the USA.

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.

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory

Evaluation of henipavirus chemical inactivation methods for the safe removal of samples from the high-containment PC4 laboratory

Henipaviruses, Hendra (HeV) and Nipah (NiV), are highly pathogenic zoonotic agents that pose a serious health risk to human life, and as such are restricted to physical containment 4 (PC4) laboratories. For further analysis of virus-infected biological specimens, it is necessary to ensure absolute inactivation of any infectious virus present before removal from the PC4 laboratory. To evaluate the inactivation of HeV and NiV within infected samples, two chemical inactivation methods were assessed. Henipavirus-infected cell monolayers treated with 4 % paraformaldehyde (PFA) showed the complete inactivation of infectious virus, with an inactivation period of 15 min resulting in more than 8-log decrease in infectious titre. NiV-infected tissue samples treated with 10 % neutral-buffered formalin (NBF) showed a complete reduction of infectious virus in 7/8 ferret organs incubated for 24 h, with the remaining tissue demonstrating complete virus inactivation after 48 h. The chemical inactivation methods described herein evaluated two simple methods of henipavirus inactivation, resulting in the complete inactivation of infectious virus – an essential requirement for the safe removal and handling of biological samples from the PC4 laboratory.

Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs.

High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.

Heat inactivation of foot-and-mouth disease virus, swine vesicular disease virus and classical swine fever virus when air-dried on plastic and glass surfaces

Foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV) and classical swine fever virus (CSFV) all cause important animal diseases. FMDV affects many different cloven-hoofed animals, whereas SVDV and CSFV are restricted to domestic and feral pigs together with wild boar. Europe is normally free of these diseases, but occasionally outbreaks happen, which can cause huge economic losses. Handling of these viruses, in particular FMDV and CSFV, is only allowed within high containment laboratories and stables. Periodically such facilities need to be decommissioned for repair or closing down, which is done by cleaning and chemical disinfection, followed by fumigation due to residual risk from virus on surfaces in inaccessible places. However, building materials in older laboratories or stables that have housed infected animals may not be well-suited for fumigation. Heat treatment is another way of inactivating viruses. In this study, we have determined the survival of infectivity in air-dried virus samples on glass and plastic surfaces incubated at room temperature or heated to 70 °C for 1, 2, 3, 5 and 7 days. Each of the tested viruses was inactivated to below the limit of detection after 24 h or 48 h of incubation at 70 °C; in contrast, some of these viruses were still infectious after 7 days of incubation at room temperature. This study provides important information that can be used in relation to decontamination of buildings and in risk-assessments.

A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

Broadly neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. Deep characterization of these bnAbs and polyclonal sera provides pivotal understanding for influenza immunity and informs effective vaccine design. However, conventional virus neutralization assays require high-containment laboratories and are difficult to standardize and roboticize. Here, we build a panel of engineered influenza viruses carrying a reporter gene to replace an essential viral gene, and develop an assay using the panel for in-depth profiling of neutralizing antibodies. Replication of these viruses is restricted to cells expressing the missing viral gene, allowing it to be manipulated in a biosafety level 2 environment. We generate the neutralization profile of 24 bnAbs using a 55-virus panel encompassing the near-complete diversity of human H1N1 and H3N2, as well as pandemic subtype viruses. Our system offers in-depth profiling of influenza immunity, including the antibodies against the hemagglutinin stem, a major target of universal influenza vaccines.