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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Principles of Biosafety

Biological safety, or biosafety, means containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. This report is a training course for Biosafety with these features and objectives: Topics associated with biological safety, including basic terms, definitions, and approaches to biological safety; regulations, consensus guidance and standards for biological safety; safety requirements for working with biological materials at Los Alamos National Laboratory (LANL); containment requirements for biological materials at LANL; and, emergency response requirements for biological materials at LANL.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2022

Principles of Biosafety (Course # 31701) [Slides]

Biological safety, or biosafety, means containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release. Containment principles, facility design, and practices & procedures that prevent occupational infections in biological research, as well as the release of the organisms to the environment, must be implemented to protect researchers, laboratory support personnel, and the environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Updated Medical Waste Management Plans and Emergency Action Plans for the Lawrence Livermore National Laboratory Main Site

Attached are the updated Medical Waste Management Plans and Emergency Action Plans for Lawrence Livermore National Laboratory’s (LLNL) Biosafety Level 2 (BSL-2) facilities and Biosafety Level 3 (BSL-3) facility. A redline version of each document has also been provided. Minor changes such as facility personnel, contact information, and titles for those that support onsite Biosafety Labs at LLNL have been made to the document. It should be noted that the most recent versions of the Medical Waste Large Quantity Generator Registration Application Form and Medical Waste Management Plan were incorporated and sourced from the Alameda County Department of Environmental Health website.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Computational Fluid Dynamics Simulations to Assess Spatial Variability and Optimal Ventilation Scenarios for Biological Laboratory Exposures

A significant amount of uncertainty exists regarding potential human exposure to laboratory biomaterials and organisms in Biosafety Level 2 (BSL-2) research laboratories. Computational fluid dynamics (CFD) modeling is proposed as a way to better understand potential impacts of different combinations of biomaterials, laboratory manipulations, and exposure routes on risks to laboratory workers. Here, in this study, we use CFD models to simulate airborne concentrations of contaminants in an actual BSL-2 laboratory under different configurations. Results show that ventilation configuration, sampling location, and contaminant source location can significantly impact airborne concentrations and exposures. Depending on the source location and airflow patterns, the transient and time-integrated concentrations varied by several orders of magnitude. Contaminant plumes from sources located near a return vent (or exhaust like a fume hood or ventilated biosafety cabinet) are likely to be more contained than sources that are further from the exhaust. Having a direct flow between the source and the exhaust (through-flow condition) may reduce potential exposures to individuals outside the air flow path. Designing a BSL-2 room with ventilation and airflow patterns that maximize through-flow conditions to the return/exhaust vents and minimize dispersion and mixing throughout the room is, therefore, recommended. CFD simulations can also be used to assist in characterizing the impacts of supply and return vent locations, room layout, and source locations on spatial and temporal contaminant concentrations. In addition, proper placement of particle sensors can also be informed by CFD simulations to provide additional characterization and monitoring of potential exposures in BSL-2 facilities.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2022

A Novel and Secure Pseudovirus Reporter System Based Assay for Neutralizing and Enhancing Antibody Assay Against Marburg Virus

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system’s credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

Bi, Jinhao↗
DOE OSTI2024

Transboundary determinants of avian zoonotic infectious diseases: challenges for strengthening research capacity and connecting surveillance networks

As the climate changes, global systems have become increasingly unstable and unpredictable. This is particularly true for many disease systems, including subtypes of highly pathogenic avian influenzas (HPAIs) that are circulating the world. Ecological patterns once thought stable are changing, bringing new populations and organisms into contact with one another. Wild birds continue to be hosts and reservoirs for numerous zoonotic pathogens, and strains of HPAI and other pathogens have been introduced into new regions via migrating birds and transboundary trade of wild birds. With these expanding environmental changes, it is even more crucial that regions or counties that previously did not have surveillance programs develop the appropriate skills to sample wild birds and add to the understanding of pathogens in migratory and breeding birds through research. For example, little is known about wild bird infectious diseases and migration along the Mediterranean and Black Sea Flyway (MBSF), which connects Europe, Asia, and Africa. Focusing on avian influenza and the microbiome in migratory wild birds along the MBSF, this project seeks to understand the determinants of transboundary disease propagation and coinfection in regions that are connected by this flyway. Through the creation of a threat reduction network for avian diseases (Avian Zoonotic Disease Network, AZDN) in three countries along the MBSF (Georgia, Ukraine, and Jordan), this project is strengthening capacities for disease diagnostics; microbiomes; ecoimmunology; field biosafety; proper wildlife capture and handling; experimental design; statistical analysis; and vector sampling and biology. Here, we cover what is required to build a wild bird infectious disease research and surveillance program, which includes learning skills in proper bird capture and handling; biosafety and biosecurity; permits; next generation sequencing; leading-edge bioinformatics and statistical analyses; and vector and environmental sampling. Creating connected networks for avian influenzas and other pathogen surveillance will increase coordination and strengthen biosurveillance globally in wild birds.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Metabolite, Protein, and Lipid Extraction (MPLEx): a method that simultaneously inactivates Middle East respiratory syndrome coronavirus and allows analysis of multiple host cell components following infection

Mass spectrometry (MS)-based, integrated proteomics, metabolomics, and lipidomics (collectively, multi-omics) studies provide a very detailed snap shot of virus-induced changes to the host following infection and can lead to the identification of novel prophylactic and therapeutic targets for preventing or lessening disease severity. Multi-omics studies with Middle East respiratory syndrome coronavirus (MERS-CoV) are challenging as the requirements of Biosafety Level 3 containment limit the numbers of samples that can be safely managed. To address these issues, the multi-omics sample preparation technique MPLEx (metabolite, protein, and lipid extraction) was developed to partition a single sample into three distinct parts (metabolites, proteins and lipids) for multi-omics analysis, while simultaneously inactivating MERS-CoV by solubilizing and disrupting the viral envelope and denaturing viral proteins. Here we describe the MPLEx protocol, highlight the step of inactivation, and describe the details of downstream processing, instrumental analysis of studies with Middle East respiratory syndrome coronavirus (MERS-CoV) are challenging as the requirements of Biosafety Level 3 containment limit the numbers of samples that can be safely managed. To address these issues, the multi-omics sample preparation technique MPLEx (metabolite, protein, and lipid extraction) was developed to partition a single sample into three distinct parts (metabolites, proteins and lipids) for multi-omics analysis, while simultaneously inactivating MERS-CoV by solubilizing and disrupting the viral envelope and denaturing viral proteins. Here we describe the MPLEx protocol, highlight the step of inactivation, and describe the details of downstream processing, instrumental analysis of the three separate analytes and their subsequent informatics pipelines.

Nicora, Carrie D.↗
DOE OSTI2024

Ongoing Cooperative Engagement Facilitates Agile Pandemic and Outbreak Response: Lessons Learned Through Cooperative Engagement Between Uganda and the United States

Pathogens threaten human lives and disrupt economies around the world. This has been clearly illustrated by the current COVID-19 pandemic and outbreaks in livestock and food crops. Here, to manage pathogen emergence and spread, cooperative engagement programs develop and strengthen biosafety, biosecurity, and biosurveillance capabilities among local researchers to detect pathogens. In this case study, we describe the efforts of a collaboration between the Los Alamos National Laboratory and the Uganda Virus Research Institute, the primary viral diagnostic laboratory in Uganda, to implement and ensure the sustainability of sequencing for biosurveillance. We describe the process of establishing this capability along with the lessons learned from both sides of the partnership to inform future cooperative engagement efforts in low- and middle-income countries. We found that by strengthening sequencing capabilities at the Uganda Virus Research Institute before the COVID-19 pandemic, the institute was able to successfully sequence SARS-CoV-2 samples and provide data to the scientific community. We highlight the need to strengthen and sustain capabilities through in-country training, collaborative research projects, and trust.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A review of experimental methods to determine bioaerosol transfer in energy recovery ventilators

Increasing ventilation is an effective method to reduce indoor airborne disease transmission. An Energy recovery ventilator (ERV) is a passive energy recovery device used to reduce the energy consumption of heating, ventilation and air-conditioning (HVAC) systems for conditioning the ventilation air. It preconditions the ventilation air by transferring energy from building exhaust air. Therefore, the ventilation and exhaust air streams interact directly/indirectly in the ERV for energy transfer. It is surmised that the ERV may transfer bioaerosols (with pathogens) from the exhaust air to ventilation air, resulting in the spread of infectious diseases. Consequently, many pandemic HVAC guidelines recommend that the use of ERVs be limited. This is a highly unsustainable direction given the increased energy requirements associated with the high ventilation provision advocated for pandemic operation. It must be noted that no validated experimental evidence exists in literature for bioaerosol transfer in ERVs. Hence, it is necessary to conduct extensive bioaerosol transfer research before adopting the unsustainable practice of limiting the utilization of ERVs. The main objective of this review study is to summarize the experimental methods and instrumentation for bioaerosol transfer research in ERVs. This comprehensive article provides a detailed overview of the generation, sampling, and analysis of bioaerosols for conducting the experiments. Further, it explains the possible mechanisms for bioaerosol transfer in various types of ERVs based on which the ERVs that need immediate attention are identified. The main contribution of this research paper is that it provides a novel experimental method which encompasses the biosafety aspects, instrumentation, performance parameters and uncertainties in conducting virus contained bioaerosol transfer study in ERVs. The findings from this review will be helpful in designing bioaerosol transfer experiments and developing future ERV test standards for such experiments.

42 ENGINEERING↗
DOE OSTI2024

A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology

Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit’s utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Phenogenomics reveals the ecology and evolution of Trichoderma fungi for sustainable agriculture

Trichoderma fungi support sustainable agriculture by suppressing plant diseases and improving crop performance. However, emerging pathogenicity of Trichoderma warrants further ecological and genetic characterization. Here we used machine learning to correlate genomic data from 37 Trichoderma strains with over 140 phenotypic traits, spanning metabolic versatility, biotic interactions, stress tolerance and reproductive strategies. We determined Trichoderma to be an ancient, genetically cohesive and physiologically diverse genus with spores capable of germination in water and dispersal via air and water droplets. Metabolic preferences indicate universal adaptation to mycoparasitism and to niches like arboreal microbial mats, alongside broader saprotrophic versatility. Our analyses are consistent with character displacement among close relatives and convergent evolution in distant lineages, with both processes shaping ecological plasticity and traits including dispersal modes, terrestrialization or endophytism. Our findings reveal that while some Trichoderma species show traits of biosafety concern, its vast ecophysiological diversity enables the development of safe, targeted bioeffectors.

Steindorff, Andrei S. [USDOE Joint Genome Institut↗
DOE OSTI2021

Near-complete depolymerization of polyesters with nano-dispersed enzymes

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme-protectant-polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme-polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. Furthermore, the results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Rapid assessment of SARS-CoV-2–evolved variants using virus-like particles

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Multiscale Airborne Infectious Disease Transmission

Airborne disease transmission is central to many scientific disciplines, including agriculture, veterinary biosafety, medicine, and public health. Legal and regulatory standards are in place to prevent agricultural, nosocomial, and community airborne disease transmission. However, the overall importance of the airborne pathway is underappreciated; e.g., the U.S. National Library of Medicine’s Medical Subjects Headings (MESH) thesaurus lacks an airborne disease transmission indexing term. This has practical consequences, as airborne precautions to control epidemic disease spread may not be taken when airborne transmission is important but unrecognized. Publishing clearer practical methodological guidelines for surveillance studies and disease outbreak evaluations could help address this situation. To inform future work, this paper highlights selected, well-established airborne transmission events, largely cases replicated in multiple, independently conducted scientific studies. Methodologies include field experiments, modeling, epidemiology studies, disease outbreak investigations, and mitigation studies. Collectively, this literature demonstrates that airborne viruses, bacteria, and fungal pathogens have the ability to cause disease in plants, animals, and humans over multiple distances, from near range (<5 m) to continental (>500 km) in scale. The plausibility and implications of undetected airborne disease transmission are discussed, including the notable underreporting of disease burdens for several airborne-transmitted diseases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species

ABSTRACT Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10 −4 , 3 × 10 −4 , and 5 × 10 −5 , respectively, in these strains using λ-Red Beta recombinase from Escherichia coli . We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Nomenclatural issues concerning cultured yeasts and other fungi: why it is important to avoid unneeded name changes

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate.

59 BASIC BIOLOGICAL SCIENCES↗