BSL-2 Plus: When and How to Implement It in the Research Environment

  |   September 14, 2018

Biosafety Level 2 Plus (BSL-2+) is the common term for laboratories where work with microorganisms is conducted in a BSL-2 laboratory with biosafety practices and procedures that are typically found at BSL-3. The need for this hybrid approach is on the rise in recent years due to increased research with viral vectors, arboviruses, and other emerging infectious diseases. But implementation can be challenging due to the fact that it is not a recognized containment level, so no clear prescription for implementation is available. Most research institutions that could benefit from this hybrid approach struggle to determine when it is safe and appropriate to use it and which BSL-3 practices to use.

BSL-2+ Safe Implementation in the Research Environment
BSL-2+ is not a recognized containment level in biosafety guidance documents such as the Centers for Disease Control and Prevention’s (CDC) Biosafety in Microbiological and Biomedical Laboratories (BMBL) or the National Institutes of Health’s (NIH) Guidelines for Recombinant and Synthetic Nucleic Acid Molecules. Other biosafety guidance documents such as the NIH’s Biosafety Considerations for Research with Lentiviral Vectors refer to “enhanced BL2 containment” (another term for BSL-2+) but do not detail how to implement it.

It is important to know that BSL-2+ is not appropriate for pathogens that are infectious via the inhalation route in the research setting. At a minimum, such pathogens must be utilized in a BSL-3 laboratory with BSL-3 practices. Clinical laboratories have different needs and challenges, for more on this you can read our post, Clinical Laboratories: Using BSL-2 Plus When Working with Pathogens Transmitted via Inhalation.

To determine if your research can be safely conducted in a BSL-2+ environment, you must conduct a risk assessment.


Critical First Step: Conducting a Risk Assessment to Identify Biosafety Level and Practices

BSL-2+ is used when a pathogen is determined to require BSL-2 physical containment using safety practices over and above the practices required at BSL-2. There is no standardized list of microorganisms, viral vectors or research projects that should be conducted at BSL-2+. To determine if BSL-2+ is suitable for your research you must first conduct a risk assessment in accordance with the process defined in the BMBL.

The risk assessment guides the selection of appropriate biosafety levels and microbiological practices, safety equipment, and facility safeguards that will contribute to preventing a laboratory exposure.

As outlined in the BMBL, the steps of the risk assessment process include:

  1. Identify agent hazards and perform an initial assessment of risk.
  2. Identify laboratory procedure hazards.
  3. Make a determination of the appropriate biosafety level and select additional precautions as indicated by the risk assessment.
  4. Evaluate the proficiencies of staff regarding safe practices and the integrity of safety equipment.
  5. Review the risk assessment with a biosafety professional, subject matter expert, and the Institutional Biosafety Committee (IBC).

The risk assessment process must be conducted for every new or revised research project. Examples of when BSL-2+ may be appropriated include:

  • Viral vectors with gene inserts consisting of oncogenes or genes of unknown function.
  • Second generation lentiviral vectors which have an increased risk in recombination to generating replication-competent lentiviruses
  • Drug resistant Risk Group Two (RG2) bacteria such as methicillin resistant Staphylococcus aureus (MRSA).
  • RG2 organisms with low infectious doses which can cause serious disease (e.g., Salmonella Typhi, Shigella)
  • Organisms where certain factors predispose individuals to infection or negative health outcomes (e.g., Zika, Listeria monocytogenes)
  • Low titer and small volumes of Human Immunodeficiency Virus (HIV), an RG3 agent.
  • High concentrations (>106 PFU/mL) of RG2 viruses.
  • Work with greater than 10 liters of a RG2 agent.
  • Organisms that present certain biocontainment and/or biosecurity concerns (e.g., low pathogenic avian influenza)

Project Review Process

The project review process is part of the risk assessment and includes these steps that must be completed before work can be approved and begin:

  1. Submission of a project registration document by the Principal Investigator (PI) to the Biosafety Officer (BSO). Define the project purpose and document the steps to be conducted with the biohazardous material.
  2. Review and discussion of the project registration document with the PI, BSO and in some cases selected members of the IBC. An IBC and adherence to the NIH Guidelines are mandatory if the institution receives federal funding and/or is located in a community with a recombinant DNA ordinance. If your institution does not require an IBC, then a similar type of review committee such as a Biosafety Committee should be part of the review process.
  3. If the review process determines that a BSL-3 containment facility is not necessary but BSL-2 practices may not provide adequate safeguards, then the use of BSL-3 practices in a BSL-2 laboratory may be appropriate. At this point a suitable BSL-2 laboratory space should be recommended (we’ll cover selecting a space in the next section) and the BSL-3 practices to be used outlined.
  4. Prior to initiation of the project the IBC members should come to consensus on the appropriate BSL-3 practices that should be applied to the proposed work. And at this stage, a suitable BSL-2 laboratory space should be agreed upon.
  5. Risk communication and training must be conducted after IBC approval and before any work is performed. The BSO should review the required BSL-3 procedures with the PI and laboratory staff. Ideally these should be documented in the form of a Standard Operating Procedure (SOP). Review the laboratory space to ensure the required BSL-2 elements are in place such as biowaste containers, sink with soap and paper towels, and certified biological safety cabinets (BSCs).


Selecting a Laboratory Space for BSL-2+

Often academic and research BSL-2 laboratories are large spaces occupied by many lab personnel working on a variety of projects and sharing lab equipment. In some cases this may not be conducive to adhering to BSL-3 practices. For this reason a separate BSL-2 laboratory space may need to be dedicated to the project that requires BSL-3 practices. Practically speaking this usually means taking a smaller BSL-2 or “tissue culture” laboratory room and dedicating it to the project. This allows access to be limited to only those people who have received the necessary training and are listed on the research protocol.

BSL-2+ Safe Implementation in the Research Environment

Selecting and Modifying BSL-3 Practices

It is important to keep in mind the BSL-3 requirements for work practices and safety equipment such as personal protective equipment (PPE) and BSCs. Sometimes the appropriate BSL-3 practices determined by the risk assessment may be limited to restricting sharps in the laboratory. In other situations, multiple BSL-3 practices are selected. Each risk assessment and project review should include a review of these practices and equipment requirements to determine the items that will protect workers and the surrounding community. Each item may be subject to discussion and there is room for adjustments, provided there is consensus among the IBC members.


Other Issues to Consider When Implementing BSL-2+ Practices

Often there are other issues that should be given careful consideration and reviewed such as:

  • If the BSL-2+ laboratory has adequate space to accommodate additional research projects, you will need to decide whether to allow other lab personnel to work in the space with materials of a lesser hazard.
  • If the project approved with BSL-3 practices involves work that occurs infrequently, you may need to decide whether to revert the lab back to a standard BSL-2 lab with BSL-2 practices. Consider the materials in use and whether the lab and equipment should be decontaminated prior to downgrading. Signage would also need to be updated. If this practice is allowed, you will need to develop an SOP detailing the process and provide training.
  • Is it appropriate for lab personnel to bring notebooks and portable electronic devices in and out of the BSL-2+ lab? Best practice is to prohibit this in order to avoid bringing contamination out of the lab, but provide other methods for information to be transmitted to the office area through a fax machine or a computer that is dedicated to the BSL-2+ lab.
  • If the BSL-2 laboratory will be renovated or built for a project using BSL-3 practices, you may find it useful to incorporate some of the BSL-3 lab facility requirements. For example, a hands-free or automatically operated sink for hand washing may be worthwhile. Or, installing an anteroom between the lab and external areas may be useful for storage of PPE.
  • If the BSL-2 laboratory is an animal biosafety level two (ABSL-2) facility, then animal biosafety practices, procedures and safety equipment criteria must be incorporated. The risk assessment guides the decision as to what ABSL-3 practices to incorporate.

The use of BSL-3 practices in a BSL-2 laboratory may be appropriate for some research projects and may contribute to the safe conduct of that research. Since there is no “one size fits all” approach, the risk assessment is key to determining whether BSL-3 practices are appropriate in a BSL-2 lab facility and what practices will be required. Strong collaboration between the PI, BSO, IBC and lab personnel is also crucial to the successful outcome.

For more information on implementing a BSL-2+, download our guide that includes illustrative case studies.


If you need help determining if a BSL-2+ is right for your research needs and how to implement it, contact us today!


In 2012, EH&E conducted a survey of academic, biotechnology and healthcare institutions to gain insight into how institutions managed implementation of BSL-3 practices and procedures in the BSL-2 environment and the drivers for using this approach. The survey results were published in an EH&E white paper originally authored by Elizabeth Gilman Duane, MS, RBP, CBSP. That paper was the basis of a published article in Applied Biosafety (cited below), which is the original source of information for this blog. In 2018 this blog was updated by Theodore A. Myatt, Sc.D., M.E.M., and Anthony J. Troiano, Jr., PH.D., RBP, of EH&E.

Gilman Duane E. 2013. A Practical Guide to Implementing a BSL-2+ Biosafety Program in a Research Laboratory. Applied Biosafety: Journal of the American Biological Safety Association, 18(1):30-36.