Poxviruses as Agents of Biological Warfare: The Importance of Ensuring Ethical Standards for Research with Viruses.

Historically, biological agents have been used to target various populations. One of the earliest examples could be the catastrophic effect of smallpox in Australia in the eighteenth century (as alleged by some historians). Modern biological techniques can be used to both create or provide protection against various agents of biological warfare. Any microorganism (viruses, bacteria, and fungi) or its toxins can be used as biological agents. Minnesota Department of Health has listed Smallpox (variola major) as a category A bioterrorism agent, even though it has been eradicated in 1980 through an extensive vaccination campaign. Category A agents are considered the highest risk to public health. Laboratory-associated outbreaks of poxviruses could cause unprecedented occupational hazards. Only two WHO-approved BSL-4 facilities in the United States and Russia are allowed to perform research on the variola virus. So, poxviruses present themselves as a classical case of a dual-use dilemma, since research with them can be used for both beneficial and harmful purposes. Although the importance of ethics in scientific research requires no further elaboration, ethical norms assume greater significance during experimentation with poxviruses. In this chapter, we will update the readers on the sensitive nature of conducting research with poxviruses, and how these viruses can be a source of potential biological weapons. Finally, specified ethical guidelines are explored to ensure safe research practices in virology.

Investigating the Potential Strategic Implications of COVID-19 for Biological Weapons Pursuit: A New Expert Simulation.

To investigate the impact of the COVID-19 pandemic on the strategic decisionmaking of leaders with respect to biological weapons, this study employed a prospective simulation technique called Asynchronous Strategic Dynamics Red Teaming. Using an immersive, multimedia simulation conducted remotely and asynchronously, the effort engaged 240 carefully selected and curated expert participants in either biological weapons or the countries of interest (as well as 60 naïve participants). Across our sample of 30 countries, simulated interest in pursuing some type of biological weapons program (defensive or offensive) remained low to moderate. While such interest increased after the simulated onset of the COVID-19 pandemic, it was limited overall, with only a handful of states showing salient increases in offensive biological weapon interest. When directly referencing why their countries might have changed their post-COVID-19 interest in biological weapons, the most commonly cited reasons were: (1) COVID-19 demonstrated the power of biological weapons to disrupt societies and cause large-scale economic harm, and (2) the pandemic revealed either the state's own or its rivals' vulnerability to diseases like COVID-19, as well as an inability to efficiently respond and contain such diseases. In sum, despite a global pandemic with massive consequences, the simulation revealed that most states are not likely to dramatically change their strategic posture regarding pursuit of offensive biological weapons.

Detection of Micrometer-Sized Virus Aerosols by Using a Real-Time Bioaerosol Monitoring System.

This study investigates a real-time handheld bioaerosol monitoring system for the detection of biological particles using UV-LED and light-induced fluorescence technology. Biological particles produce both scattering and fluorescence signals simultaneously, which can help distinguish them from general particles. The detected scattering, fluorescence, and simultaneous signals are then converted into photon signals and categorized based on predetermined criteria. A reliable biological particle generator was required to validate the performance of the system. This study explores the use of an M13 bacteriophage as a virus simulant of biological agents and employs a customized inkjet aerosol generator to produce M13 bacteriophage aerosols of a specific size by controlling the concentration of M13. We confirmed that micro-sized, narrowly dispersed M13 aerosols were efficiently generated. Additionally, we confirmed the performance of this real-time handheld bioaerosol monitoring system by detecting viruses.

Plasmids Increase the Competitive Ability of Plasmid-Bearing Cells Even When Transconjugants Are Poor Donors, as Shown by Computer Simulations.

Bacterial cells often suffer a fitness cost after conjugative plasmids' entry because these cells replicate slower than plasmid-free cells. Compensatory mutations may appear after tens of or a few hundred generations, reducing or eliminating this cost. A previous work based on a mathematical model and computer simulations has shown that plasmid-bearing cells already adapted to the plasmid may gain a fitness advantage when plasmids transfer into neighboring plasmid-free cells because these cells are still unadapted to the plasmid. These slow-growing transconjugants use fewer resources, which can benefit donor cells. However, opportunities for compensatory mutations in transconjugants increase if these cells become numerous (through replication or conjugation). Moreover, transconjugants also gain an advantage when transferring the plasmid, but the original donors may be too distant from conjugation events to gain an advantage. To understand which consequence prevails, we performed further computer simulations allowing versus banning transfer from transconjugants. The advantage to donors is higher if transconjugants do not transfer plasmids, mainly when donors are rare and when the plasmid transfer rate (from donors) is high. These results show that conjugative plasmids are efficient biological weapons even if the transconjugant cells are poor plasmid donors. After some time, conjugative plasmids gain other host-benefit genes, such as virulence and drug-resistance.

ASSESSMENT OF THE CONSEQUENCES OF THE DETERIORATION OF THE EPIDEMIOLOGICAL SITUATION DURING HOSTILITIES.

OBJECTIVE: The aim: Scientific substantiation of the methodology for predicting the consequences of the worsening of the epidemic situation on the territory of Ukraine during military operations for the timely adoption of measures for the medical protection of military personnel in conditions of biological contamination. PATIENTS AND METHODS: Materials and methods: Determination and generalization of the impact of biological contamination due to the use of biological weapons were carried out considering the main determinants of the epidemic process using the index and coefficient of medical protection. Applied methods of scientific research: epidemiological, system, and information approach. RESULTS: Results: The authors proposed indicators that consider the pathogenicity of the infectious agent, contagiousness, the degree of non-specific protection of servicemen, specific protection of servicemen, and the sanitary-epidemiological state of the area of operations of troops (forces). Relevant epidemic situations were simulated, and the index and coefficient of medical protection were calculated to predict the consequences of the worsening of the epidemic situation to make timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination during the repulsion of armed aggression. CONCLUSION: Conclusions: In the conditions of biological contamination, when biological weapons and biological terrorism are used, the epidemic process in the army is intensified, which requires timely decisions regarding the implementation of medical protection measures for military personnel in conditions of biological contamination.

[The role of the pulmonologist in an armed conflict]. / Place du pneumologue en situation de conflit armé.

INTRODUCTION: Recent news points to the eventuality of an armed conflict on the national territory. STATE OF THE ART: In this situation, pulmonologists will in all likelihood have a major role to assume in caring for the injured, especially insofar as chest damage is a major cause of patient death. PERSPECTIVES: The main injuries that pulmonologists may be called upon to treat stem not only from explosions, but also from chemical, biological and nuclear hazards. In this article, relevant organizational and pedagogical aspects are addressed. Since exhaustiveness on this subject is unattainable, we are proposing training on specific subjects for interested practitioners. CONCLUSION: The resilience of the French health system in a situation of armed conflict depends on the active participation of all concerned parties. With this in mind, it is of prime importance that the pneumological community be sensitized to the potential predictable severity of war-related injuries.

Assessing emerging technologies from an arms control perspective.

Developments in science and technology improve health and wellbeing of humankind, for example with better methods to detect and treat diseases. However, some advances have led to the development of weapons of mass destruction: chemical and biological weapons. Although banned by international treaties, chemical weapons have been used in recent years in assassinations and the Syrian civil war. Additionally, biological weapons became the subject of recent suspicions and allegations. While not limited to these fields, the so-called dual-use potential-the possibility to apply aspects both with benevolent or malevolent intentions-is especially pronounced in the life sciences. Here, we showcase some areas explored at the conference series Spiez CONVERGENCE that facilitates an exchange between science, arms control and international security. Together, these communities discuss the potential impact of life scientific advances on the Chemical and Biological Weapons Conventions. Enabled by digital technologies, DNA sequencing and synthesis provide the toolbox to (re)construct viruses and cells, which demonstrated invaluable during the COVID-19 pandemic but bear the misuse risk to allow intentionally triggering an outbreak. Open databases and algorithms could be used to generate new chemical weapons. We argue that preventing unintended consequences of life science research while promoting its benefits with responsible science, requires awareness and reflection about unexpected risks of everyone involved in the research process. The strength of the ban of chemical and biological weapons also depends on scientists interacting with policy makers in evaluating risks and implementing measures to reduce them.

Peptide Toxins as Biothreats and the Potential for AI Systems to Enhance Biosecurity.

Biological weapons have been used for thousands of years, but recent advances in synthesis technologies have made peptide and protein toxin production more accessible and pose a threat to biosecurity worldwide. Natural toxins such as conotoxins, certain hemolytic compounds, and enterotoxins are peptide agents that can be synthesized in an environment with weak biosecurity measures and rudimentarily weaponized for limited use against smaller targets for lethal or nonlethal effects. Technological advances are changing the threat landscape around biological weapons and potentially facilitating a shift from state sponsored to more micro-level threats stemming from terror cells, insider threats, and lone wolf attacks. Here, we present the reader with an overview of the threat of peptide and protein toxins, provide examples of potent peptide toxins, and introduce capabilities of a proposed biosecurity program utilizing artificial intelligence that unifies commercial nucleotide and peptide synthesis vendors.

Systematic approach towards establishing a National Inventory of Dangerous Pathogens.

International regulations stipulate that countries need to organize their biosafety and biosecurity systems to minimize the risk of accidental (biosafety) or malicious intentional (biosecurity) release of dangerous pathogens. International Health Regulations (IHR) benchmarks from the WHO state that even for a level of limited capacity countries need to 'Identify and document human and animal health facilities that store/maintain dangerous pathogens and toxins in the relevant sectors and health professionals responsible for them'. This study provides a stepwise, systematic approach and best practices for countries to initiate a national inventory of dangerous pathogens. With a national inventory of dangerous pathogens a country can identify and document information in a dedicated electronic database on institutes that store or maintain dangerous pathogens. The systematic approach for the implementation of a national inventory of dangerous pathogens consists of four stages; identification, preparation, implementation, and maintenance and evaluation. In the identification phase, commitment of the relevant national ministries is to be established, and a responsible government entity needs to be identified. In the preparatory phase, a list of pathogens to be incorporated in the inventory, as well as a list of institutes to include, is to be agreed upon. In the implementation phase, the institutes are contacted, and the collected data is stored safely and securely in a electronical database. Finally, in the maintenance and evaluation phase meaningful insights are derived and reported to the relevant government authorities. Also, preparations for updates and modifications are undertaken, such as modifications of pathogen lists or institute lists. The approach and database, which is available from the authors, have been tested for the implementation of a national inventory of dangerous pathogens in multiple East-African countries. A national inventory of dangerous pathogens helps countries in strengthening national biosafety and biosecurity as well as in their compliance to IHR.

Assessing the Risks and Benefits of Advances in Science and Technology: Exploring the Potential of Qualitative Frameworks.

Continuing rapid advances in science and technology both pose potential risks and offer potential benefits for the effective implementation of the Biological Weapons Convention (BWC). The lack of commonly accepted methods for assessing relevant risks and benefits present significant challenges to building common understandings that could support policy choices. This article argues that qualitative frameworks can provide the basis to structure BWC discussions about potential risks and benefits, reveal areas of agreement and disagreement, and provide a basis for continuing dialogue. It draws on the results of a workshop held in Geneva during the 2019 BWC Meetings of Experts. A diverse group of international experts were given the opportunity to apply 2 qualitative frameworks developed specifically to assess potential biosecurity concerns arising from emerging science and technology to BWC-relevant case examples. Participants discussed how such frameworks might be adapted and put into action to help support the BWC. They also began a discussion of how a comparable framework to assess potential benefits could be developed.

Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model.

The purpose of the current work was to utilize a three dimensional (3D) corneal epithelial tissue model to study dry eye disease and oxidative stress-related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. 3D corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, a decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared the effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of damage. The topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.

Characterization of Cholinesterases From Multiple Large Animal Species for Medical Countermeasure Development Against Chemical Warfare Nerve Agents.

Organophosphorus (OP) compounds, which include insecticides and chemical warfare nerve agents (CWNAs) such as sarin (GB) and VX, continue to be a global threat to both civilian and military populations. It is widely accepted that cholinesterase inhibition is the primary mechanism for acute OP toxicity. Disruption of cholinergic function through the inhibition of acetylcholinesterase (AChE) leads to the accumulation of the neurotransmitter acetylcholine. Excess acetylcholine at the synapse results in an overstimulation of cholinergic neurons which manifests in the common signs and symptoms of OP intoxication (miosis, increased secretions, seizures, convulsions, and respiratory failure). The primary therapeutic strategy employed in the United States to treat OP intoxication includes reactivation of inhibited AChE with the oxime pralidoxime (2-PAM) along with the muscarinic acetylcholine receptor antagonist atropine and the benzodiazepine, diazepam. CWNAs are also known to inhibit butyrylcholinesterase (BChE) without any apparent toxic effects. Therefore, BChE may be viewed as a "bioscavenger" that stoichiometrically binds CWNAs and removes them from circulation. The degree of inhibition of AChE and BChE and the effectiveness of 2-PAM are known to vary among species. Animal models are imperative for evaluating the efficacy of CWNA medical countermeasures, and a thorough characterization of available animal models is important for translating results to humans. Thus, the objective of this study was to compare the circulating levels of each of the cholinesterases as well as multiple kinetic properties (inhibition, reactivation, and aging rates) of both AChE and BChE derived from humans to AChE and BChE derived from commonly used large animal models.

What Electrochemical Biosensors Can Do for Forensic Science? Unique Features and Applications.

This article critically discusses the latest advances in the use of voltammetric, amperometric, potentiometric, and impedimetric biosensors for forensic analysis. Highlighted examples that show the advantages of these tools to develop methods capable of detecting very small concentrations of analytes and provide selective determinations through analytical responses, without significant interferences from other components of the samples, are presented and discussed, thus stressing the great versatility and utility of electrochemical biosensors in this growing research field. To illustrate this, the determination of substances with forensic relevance by using electrochemical biosensors reported in the last five years (2015-2019) are reviewed. The different configurations of enzyme or affinity biosensors used to solve analytical problems related to forensic practice, with special attention to applications in complex samples, are considered. Main prospects, challenges to focus, such as the fabrication of devices for rapid analysis of target analytes directly on-site at the crime scene, or their widespread use and successful applications to complex samples of interest in forensic analysis, and future efforts, are also briefly discussed.

Intramuscular administration of hexachloroplatinate reverses cyanide-induced metabolic derangements and counteracts severe cyanide poisoning.

Cyanide is a highly toxic industrial chemical that is widely used by manufactures. Smoke inhalation during household fires is the most common source of cyanide poisoning while additional risks to civilians include industrial accidents and terrorist attacks. Despite the risks to large numbers of individuals, an antidote capable of administration at scale adequate for a mass casualty, prehospital scenario does not yet exist. Previously, we demonstrated that intravenous cisplatin analogues accelerate recovery from cyanide poisoning in mice and rabbits. Of the dozens of platinum-based organometallic complexes tested, hexachloroplatinate (HCP) emerged as a promising lead compound, exhibiting strong affinity for cyanide and efficacy across model systems. Here, we show HCP is an antidote to lethal cyanide exposure and importantly is effective when delivered intramuscularly. The pharmacokinetic profile of HCP exhibited bioavailability in the systemic circulation 2.5 minutes post-treatment and subsequent renal clearance of HCP-cyanide. HCP restored parameters of cellular physiology including cytochrome oxidase redox state and TCA cycle metabolism. We next validated these findings in a large animal model (swine). Finally, preclinical safety studies in mice revealed minimal toxicity. Cumulatively, these findings demonstrate hexachloroplatinate is a promising lead compound for development of an intramuscular injectable cyanide antidote for mass casualty scenarios.

Establishment of a National Inventory of Dangerous Pathogens in the Republic of Uganda.

One of the challenges of global biosecurity is to protect and control dangerous pathogens from unauthorized access and intentional release. A practical and feasible option to protect life science institutes against theft and sabotage, and secure their biological materials against misuse, is to establish a national electronic database with a comprehensive overview of the locations of all controlled dangerous pathogens in a country. This national database could be used as an instrument to secure and account for dangerous pathogens in a country, but it could also assist in establishing a biosecurity assessing and monitoring system for laboratories that work with these controlled biological agents. The Republic of Uganda is one of the first countries, prompted by the World Health Organization's (WHO's) Joint External Evaluation (JEE), to implement a national electronic database that assembles information collected from relevant Ugandan laboratories. This Ugandan Inventory of Dangerous Pathogens is different from an institute-specific pathogen inventory system, as it is intended to store the information collected from laboratories in the country working with dangerous pathogens in 1 centralized secure location. The Uganda National Council for Science and Technology (UNCST) has coordinated the implementation of the Ugandan national inventory. The inventory was recognized by the WHO JEE as contributing to Uganda's developed capacities regarding biosafety and biosecurity. This article describes the steps in implementing Uganda's National Inventory of Dangerous Pathogens. In addition, it presents a straightforward approach that can be adapted by other countries that aim to enhance their biosecurity capacities.

A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS.

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

Development of a Biosecurity Checklist for Laboratory Assessment and Monitoring.

INTRODUCTION: Laboratory biosecurity is of continuously growing interest due to increasing concerns about deliberate misuse of biological materials and emerging biological risks. These risks continue to be magnified by globalization, the rapid pace of scientific development, and dual-use technologies. Worldwide laboratory capacities are expanding, which calls for concrete actions to improve laboratory biosafety and biosecurity practices to protect researchers and the community. Hence, laboratories require comprehensive biorisk management programs to minimize the risk of accidental and deliberate release of infectious biological materials. OBJECTIVE: Malaysia has prioritized the concern of national biosecurity and aims to consolidate laboratory biosecurity performance to detect and prevent the deliberate release of biological agents. METHODS: Two 3-day workshops were organized over the course of four months in which Malaysia collaborated with The Netherlands. This bilateral engagement aimed to integrate biosecurity practices in their national biorisk management programs, and resulted into a comprehensive biosecurity checklist for laboratory assessment and monitoring. RESULTS: This biosecurity checklist is based on Malaysian and Dutch expert opinions and national and international guidelines and regulations. The biosecurity checklist is a survey-driven tool that consists of a set of concrete questions for each key biosecurity area, which are discussion points for assessment. CONCLUSION: We display a practical biosecurity checklist for laboratory assessment and monitoring. Although the presented checklist was the template for the specific Malaysia checklist, it could serve as a template for other countries.