Viral Replicon Systems and Their Biosafety Aspects.

Introduction: Viral RNA replicons are self-amplifying RNA molecules generated by deleting genetic information of one or multiple structural proteins of wild-type viruses. Remaining viral RNA is used as such (naked replicon) or packaged into a viral replicon particle (VRP), whereby missing genes or proteins are supplied via production cells. Since replicons mostly originate from pathogenic wild-type viruses, careful risk consideration is crucial. Methods: A literature review was performed compiling information on potential biosafety risks of replicons originating from positive- and negative-sense single-stranded RNA viruses (except retroviruses). Results: For naked replicons, risk considerations included genome integration, persistence in host cells, generation of virus-like vesicles, and off-target effects. For VRP, the main risk consideration was formation of primary replication competent virus (RCV) as a result of recombination or complementation. To limit the risks, mostly measures aiming at reducing the likelihood of RCV formation have been described. Also, modifying viral proteins in such a way that they do not exhibit hazardous characteristics in the unlikely event of RCV formation has been reported. Discussion and Conclusion: Despite multiple approaches developed to reduce the likelihood of RCV formation, scientific uncertainty remains on the actual contribution of the measures and on limitations to test their effectiveness. In contrast, even though effectiveness of each individual measure is unclear, using multiple measures on different aspects of the system may create a solid barrier. Risk considerations identified in the current study can also be used to support risk group assignment of replicon constructs based on a purely synthetic design.

Biosafety and Biosecurity in Containment: A Regulatory Overview.

When biosafety for contained use is addressed in international fora and discussions, often the topic is limited to working with genetically modified organisms (GMOs) in facilities such as laboratories, animal facilities, and greenhouses. However, the scope of biosafety in containment encompasses many other types of biological materials, such as human, animal and plant pathogens, nucleic acids, proteins, human samples, animals or plants, or by-products thereof, and overlaps often with the topic of biosecurity. This is also reflected in the regulations that apply for activities with biological materials in contained facilities. The common denominator of these regulations is the focus on protection of people and environment, while applying the key principles of risk assessment and risk management. This review provides an overview of regulatory frameworks for biosafety and biosecurity in containment around the globe, as well as points out overlap with other regulatory frameworks, such as the Nagoya Protocol, or Plant and Animal Health regulations.

Poliopolis: pushing boundaries of scientific innovations for disease eradication.

Although global polio eradication is within reach, sustained eradication of all polioviruses requires cessation of oral poliovirus vaccine use to mitigate against vaccine-derived poliovirus circulation and vaccine-associated paralytic poliomyelitis. The first step in this direction was the WHO-recommended global withdrawal of live attenuated type 2 Sabin poliovirus from routine immunisation in May 2016, with future use restricted to outbreak response, and handling controlled by strict containment provisions (GAPIII). This creates unique challenges for development and testing of novel type 2 poliovirus vaccines. We describe the creation of a novel purpose-built containment facility, Poliopolis, to study new monovalent OPV2 vaccine candidates in healthy adult volunteers, which may be a model for future endeavors in vaccine development for emergency use.

Biotechnologically produced chitosan for nanoscale products. A legal analysis.

Conventionally, chitosans are derived from shrimp and other crustacean shells. Biotechnology offers an alternative route to produce chitosans and more importantly, specific chitosan structures tailored to the needs of a diversity of industries. However, for biotech chitosans and products thereof to be commercialised, legislation should not create a burden. Here, the requirements of the EU regulatory framework have been analysed for the entire chain from research to development and production of several potential applications including nanomaterials. The animal or biotechnological origin leads to specific requirements in production of the raw material. No EU legislation dedicated to nanomaterials has been adopted. Instead, products are governed under the respective existing product legislation subject to extra requirements for safety assessment. While a knowledge gap exists on hazards related to nanomaterials in general, there is a need to establish realistic regulatory study designs to assess the safety of specific products. Furthermore, as many of the existing chitosan applications are not considered nanomaterials, it would be discriminatory to treat biotechnology derived products differently.

A Curriculum-Based Approach to Teaching Biosafety Through eLearning.

Anyone working in biosafety capacity enhancement faces the challenge of ensuring that the impact of a capacity enhancing activity continues and becomes sustainable beyond the depletion of funding. Many training efforts face the limitation of one-off events: they only reach those people present at the time. It becomes incumbent upon the trainees to pass on the training to colleagues as best they can, whilst the demand for the training never appears to diminish. However, beyond the initial effort to establish the basic content, repeating capacity enhancement events in different locations is usually not economically feasible. Also, the lack of infrastructure and other resources needed to support a robust training programme hinder operationalizing a "train-the-trainer" approach to biosafety training. One way to address these challenges is through the use of eLearning modules that can be delivered online, globally, continuously, at low cost, and on an as-needed basis to multiple audiences. Once the modules are developed and peer-reviewed, they can be maintained on a remote server and made available to various audiences through a password-protected portal that delivers the programme content, administers preliminary and final exams, and provides the administrative infrastructure to register users and track their progress through the modules. Crucial to the implementation of such an eLearning programme is an approach in which the modules are intentionally developed together as a cohesive curriculum. Once developed, such a curriculum can be released as a stand-alone programme for the training of governmental risk assessors and regulators or used as accredited components in post-graduate degree programmes in biosafety, at minimal cost to the government or university. Examples from the portfolio of eLearning modules developed by the International Centre for Genetic Engineering and Biotechnology (ICGEB) are provided to demonstrate these key features.

Off-Patent Transgenic Events: Challenges and Opportunities for New Actors and Markets in Agriculture.

More than 20 years ago, the first genetically modified (GM) plants entered the seed market. The patents covering the first GM plants have begun to expire and these can now be considered as Off-Patent Events. Here we describe the challenges that will be faced by a Secondary Party by further use and development of these Off-Patent Events. Indeed, the conditions for Off-Patent Events are not available yet to form the basis for a new viable industry similar to the generic manufacturers of agrochemicals or pharmaceutical products, primarily because of (i) unharmonized global regulatory requirements for GM organisms, (ii) inaccessibility of regulatory submissions and data, and (iii) potential difficulties to obtain seeds and genetic material of the unique genotypes used to generate regulatory data. We propose certain adaptations by comparing what has been done in the agrochemical and pharmaceutical markets to facilitate the development of generics. Finally, we present opportunities that still exist for further development of Off-Patent Events in collaboration with Proprietary Regulatory Property Holders in emerging markets, provided (i) various countries approve these events without additional regulatory burdens (i.e., acceptance of the concept of data transportability), and (ii) local breeders agree to meet product stewardship requirements.

Use of RODAC plates to measure containment of Mycobacterium tuberculosis in a Class IIB biosafety cabinet during routine operations.

OBJECTIVE/BACKGROUND: Guidelines for the manipulation of Mycobacterium tuberculosis (MTB) cultures require a Biosafety Level 3 (BSL-3) infrastructure and accompanying code of conduct. In this study, we aimed to validate and apply detection methods for viable mycobacteria from surfaces in a BSL-3 MTB laboratory. METHODS: We evaluated phenotypic (Replicate Organism Detection and Counting [RODAC] plates) and molecular (propidium monoazide [PMA]-based polymerase chain reaction [PCR]) approaches for the detection of viable mycobacteria, as well as the effect of 70% ethanol applied for 5min for disinfection against mycobacteria. For validation of the method, recovery of serial dilutions of Mycobacterium bovis bacillus Calmette-Guérin from glass slides was measured. Subsequently, we stamped surfaces in and around the biosafety cabinet (BSC) after different technicians had manipulated high bacterial load suspensions for routine drug-susceptibility testing in a Class II BSC. RESULTS: RODAC stamping could detect as few as three bacteria on slides stamped either 5min or 60min after inoculation. PMA-based PCR, tested in parallel, did not pass validation. Mycobacteria were still detected after 5-min disinfection with ethanol 70%. In the BSL-3, from 201 RODAC-stamped surfaces, MTB was detected in four: three inside a BSC-on a tube cap and on an operator's gloves-and one outside, on an operator's gown. CONCLUSION: RODAC plates detect mycobacteria at low numbers of microorganisms. In addition, this method allowed us to show that 70% ethanol does not reliably kill mycobacteria when applied for 5min to a dried surface, and that MTB bacilli may arrive outside a Class II BSC during routine practice, although the route could not be documented.