WORKING WITH BIOLOGICAL MATERIALS

Viral vectors are common tools used in molecular biology as a delivery system for genetic materials into cells that may normally be difficult to transfect. Viral vectors are constructed by replacing wild type viral genes with transgenes of interest. This usually involves removal of viral replication genes so that the vector is rendered safer than the wild-type virus (i.e., viral vector is replication incompetent). While viral vectors are modified in such a way as to minimize the risks when handling them there still may be biosafety concerns associated with their use. These include:

• Replication competence - While these vectors are designed to be replication deficient, it is impossible to completely control for the possibility of the generation of replication competent virus or reversion to wild type virus through recombination.
• Off target effects - For viral vectors that integrate into the host cell genome, off target effects such as insertional mutagenesis can be a safety concern. Integration into genes that are important for cell division or programmed cell-death can potentially result in oncogene activation or tumor-suppressing gene inactivation.
• Transgene hazards - The gene of interest can present a hazard itself. Transgenes that are known for oncogenic potential, encode apoptotic or toxin molecules present a higher biosafety risk and should be evaluated during the initial risk assessment prior to beginning work with these materials.

Viral vectors used in research involving recombinant techniques are regulated by the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (rsNAM). Project directors planning to conduct research with viral vectors and rsNAM must submit their research protocols to the Institutional Biosafety Committee (IBC) for review. For further information please visit the Office of Research Compliance website for biosafety.

The following are commonly used viral vectors along with required containment levels and other related safety information:
Adeno-Associated Viruses (AAV)
Adeno-associated viruses are small, non-enveloped viruses with single strand linear DNA. They are members of the Parvoviridae family and there are 12 serotypes. To replicate, AAV require helper viruses (wt adenovirus or herpesvirus). In the absence of these helper viruses, AAV can stably integrate into the host cell genome. AAV vectors are non-pathogenic and can infect dividing and non-dividing cells making them preferred viral vectors for many applications.

• Potential health hazards - While there are no known diseases associated with AAV human serotype, insertional mutagenesis is a theoretical possibility due to the ability of AAV to integrate into the host cell genome.
• Laboratory hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols.
• Risk Group - Risk Group1
• Containment - BSL-1/ABSL-1 (Exceptions include presence of a helper virus then BSL-2 /ABSL-2; Packaging in human cell lines then BSL-2; Potentially hazardous transgenes such as oncogenes and toxins BSL-2/ABSL-2)
• Disinfection/decontamination - 0.5-1% sodium hypochlorite (10% Bleach), 2% glutaraldehyde, 0.25% sodium dodecyl sulfate, Autoclave (121°C, 1hr)

Adenoviruses
Adenoviruses are medium-sized, non-enveloped viruses with double-stranded linear DNA. They are members of the Adenoviridae family and there are close to 100 different serotypes with over 40 being human pathogens. Adenoviral vectors are non-integrating and are a popular choice in gene therapy due to their high transduction efficiencies, ability to infect many cell types, and high level of transgene expression.

• Potential health hazards - Exposure to adenovirus can cause a wide range of symptoms including bronchitis, sore throat, fever, diarrhea and pink eye. Infections can be more severe in very young and immunocompromised individuals.
• Laboratory hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols
• Risk group - Risk Group 2
• Containment - BSL-2/ABSL-2. Adenovirus must be administered to animals under ABSL-2 containment. Animals must be housed under ABSL-2 containment for 72 hours after adenovirus administration, after which animals may be moved to ABSL-1 containment.
• Disinfection/decontamination - 1% sodium hypochlorite (10% Bleach), 2% glutaraldehyde, 0.25% sodium dodecyl sulfate, Autoclave (121°C, 1hr)

Lentiviruses
Lentiviruses are enveloped retroviruses that are characterized by a long incubation period, immune evasion, and persistent infections in their natural hosts. They have single stranded and linear RNA that is reverse transcribed to produce DNA upon entry into the host cell. This DNA transcript then integrates into the host's genome. Lentiviruses have the ability to integrate into the genome of non-dividing cells, a feature that distinguishes lentiviruses from other retroviruses. Adverse events can include leukemogenesis and oncogenesis through insertional mutagenesis.There are five recognized serotypes with HIV-derived vectors being the most commonly used lentiviruses in biomedical research.

• Potential health hazards - Infection with Lentivirus (HIV) can cause initial symptoms that are flu-like. Symptoms can be more severe in very young and immunocompromised individuals.  Infection is persistent and lifelong due to their ability to integrate into the host chromosome and the ability to evade host immunity.
• Laboratory hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites) and ingestion. Aerosol transmission unknown. Major risks associated with HIV-1 based lentivirus vectors are the potential for generation of replication-competent lentivirus and potential for oncogenesis.
• Risk group - Risk Group 3
• Containment - BSL-2/ABSL-2. Lentivirus must be administered to animals under ABSL-2 containment. Animals must be housed under ABSL-2 containment for 72 hours after adenovirus administration, after which animals may be moved to ABSL-1 containment.
• Disinfection/decontamination - 0.5% - 1% sodium hypochlorite (10% Bleach), to a lesser extent 70% ethanol, UV light, pH higher or lower than the optimal level of 7.1, Autoclave (121°C, for at least 30 minutes)

Rotovirus / Moloney Murine Leukemia Viruses (MMLV)
Moloney murine leukemia virus (MMLV) is an enveloped single-stranded RNA gammaretrovirus that can cause cancer in mice. MMLV can randomly integrate into the genome of a host cell. The host range of recombinant MMLV depends on the specificity of the viral envelope. Ecotrophic vectors can only infect rodent cells whereas amphotrophic vectors can potentially infect a wide range of cell types.  

• Potential health hazards - Pseudotyped MMLV that infect human cells can result in insertional mutagenesis that can lead to development of malignancies.
• Laboratory hazards - Mucus membrane exposure, contact with tissues and bodily fluids of animals, and parenteral inoculation (e.g. needle sticks and animal bites)
• Risk group - Risk Group 1 (Ecotrophic), Risk Group 2 (Amphotrophic or pseudotyped)
• Containment - EcotropicMMLV  : BSL-1/ABSL-1.  Amphotropic or VSV-g pseudotyped vectors containing biological toxin or gene with oncogenic potential: BSL-2/ABSL-2
• Disinfection/decontamination - 0.5% - 1% sodium hypochlorite (10% Bleach), 70% ethanol, 2% glutaraldehyde

Baculovirus
Baculovirus is a lytic, enveloped DNA virus that infects insects. Baculovirus vectors are mainly used to produce high levels of recombinant proteins in insect cells as they have large cloning capacity and low cytotoxicity. Wild type virus is non-pathogenic to humans but recombinant virus using specific promoters allows for gene expression in mammalian cell lines.

• Potential health hazards - Certain transgenes (e.g. oncogenes, mammalian specific toxins) could potentially pose a health risk and must be taken into consideration when performing an initial risk assessment.
• Laboratory hazards - Direct contact, droplet exposure of the mucus membranes, parenteral inoculation
• Risk group - Risk Group 1
• Containment - BSL-1/ABSL-1. Baculovirus pseudotyped with VSV-g can transduce human cell line s and should be handled at BSL-2/ABSL-2.
• Disinfection/decontamination - 10% Bleach, 70% ethanol, Autoclave (121°C, for at least 30 minutes)

Rabies virus
Rabies virus is a neurotropic virus in the Rhabdoviridae family of viruses. It causes a common zoonotic infection from bats and other wild animals that results in encephalitis or paralysis and is often fatal. Its neuronal tropism makes it a good candidate for studying neuronal trafficking or to express genes efficiently in neurons.

• Potential health hazards - Infection with rabies virus can cause Flu-like symptoms, gastrointestinal, neurological and respiratory symptoms. The virus can cause acute progressive encephalomyelitis. Once clinical signs of rabies infection manifest themselves, the disease cannot be cured or treated and is nearly always fatal.
• Laboratory hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols
• Risk group - Risk Group 2 (Recombinant vector). Rabies virus is Risk Group 3.
• Containment - BSL-2/ABSL-2 for most constructs but may require BSL-3/ABSL-3 depending on risk assessment
• Disinfection/decontamination - 70% ethanol, phenol, formalin, ether, trypsin, some other detergents, UV light

Biological toxins encompass a vast range of peptides, small molecules, and macromolecular proteins that cause disease by interfering with biological processes. They are produced by living organisms, are unable to replicate, and do not result in communicable diseases. Many biological toxins have been evolutionarily optimized to rapidly disrupt critical biological functions at low concentrations. Biological toxins remarkable combination of specificity and potency has resulted in widespread use for clinical and research purposes.

Laboratory workers can be exposed to biological toxins through a variety of routes, including inhalation of powders, aerosols, or volatile substances; ingestion; injection; and absorption through dermal, mucosal or ocular tissues. Due to their potency, internalization of even small doses may result in death or severe incapacitation. It is critically important for those working with biological toxins to understand and implement appropriate laboratory safety principles.

Each laboratory worker that plans to work with toxins of biological origin must be trained prior to beginning work with toxins. This training must include awareness of the risks associated with the specific toxins to be used including risks associated with transfer of solubilized toxins, manipulation of waste solutions, contamination of materials and equipment, and decontamination after routine operations and spills.

Guidelines for Biological Toxin Use 

• An inventory control system should be established and audited on a regular basis to account for toxin quantity, use, and disposition.
• Toxins should be stored in containers that clearly list the toxin contents, points of contact for responsible laboratory staff, and emergency contacts.
• The use of locks on storage containers/locations offers additional control over toxin access and is highly recommended.
• Work with toxins should only be done in designated rooms or areas with pre-determined bench areas.
• When toxins are in use, the area should have clearly posted signage indicating that toxins are in use, minimum requirements for PPE, and emergency points of contact.
• Treatment plans for accidental exposures should be prepared and available to all laboratory staff and emergency responders.

Safety Equipment and Containment
Routine operations with dilute toxin solutions are conducted under BSL-2 containment with the aid of PPE (laboratory coat, eye protection, gloves) and a well-maintained BSC, chemical fume hood or comparable engineering controls. Toxin(s) should be removed from the hood or BSC only after the exterior of the closed primary container has been decontaminated and placed in a clean secondary container. The interior of the hood or BSC should be decontaminated periodically (i.e. at the end of the day or after a spill). Selected operations with toxins may require additional precautions and potentially the use of BSL-3 containment. This will be determined by a thorough Risk Assessment and consultation with biosafety staff.

Additional Precautions
Many biological toxins are highly potent, and emphasis must be placed on evaluating and modifying experimental procedures to avoid inadvertent generation of toxin aerosols.

• Centrifugation of cultures or materials potentially containing toxins should only be performed using sealed, thick-walled tubes in safety centrifuge cups or sealed rotors.
• Sealed centrifuge safety cups or sealed rotor should be taken from the centrifuge to a BSC (or comparable engineering control) prior to breaking the seal and removing centrifugation tubes.
• Experiments should be planned to eliminate or minimize work with dry toxin or toxin-containing formulations. Unavoidable operations with dry toxins should only be undertaken with appropriate respiratory protection and engineering controls.
• Animal work involving aerosol exposure should be conducted in a certified Class III BSC or similar containment device.
• Vaccinations against some biological toxins are available and may be appropriate for laboratory workers.

Decontamination 
Decontamination of a biological toxin(s) means the toxin is rendered inactive and is no longer capable of exerting its toxic effect. Toxin stability varies considerably outside of physiological conditions depending on temperature, pH, ionic strength, presence of co-factors, and other characteristics of the surrounding matrix.

Most toxins are susceptible to steam inactivation (121°C for one hour) or to chemical inactivation with dilute sodium hydroxide (NaOH) at concentrations of 0.1- 0.25 N, and/or sodium hypochlorite (NaOCl) solutions at concentrations of 0.1% - 2.5.% (w/v). However, inactivation procedures should not be assumed to be 100% effective without validation using specific toxin bioassays. Additional information related to decontamination of selected toxins can be found in Appendix I of the Biosafety in Microbiological and Biomedical Laboratories (BMBL, 6th ed.).

Spills
In the event of a liquid spill:

• Avoid splashes or generating aerosols during clean-up by covering the spill with dry paper towels of other disposable, absorbent material.
• Ensure that appropriate PPE (Lab coat, gloves, eye protection) is worn during the clean-up.
• Apply an appropriate decontamination solution to the spill, beginning at the perimeter and working towards the center.
• Allow sufficient contact time for the decontamination solution to inactivate the toxin.
• Restrict access to the contaminated area until the decontamination is complete.

In the event of toxin powder:

• PPE should include respiratory protection in addition to PPE used for liquid spills.
• For a spill within the BSC, gently cover the powder spill with damp absorbent paper towels to avoid raising dust than follow the steps described for a liquid spill.
• For a spill outside of the BSC, the area should be immediately evacuated, and the area restricted until the area has been thoroughly decontaminated (this may include turning off the HVAC system or removing and discarding HVAC filters that may have been contaminated).

Select Toxins
HHS and the USDA have identified a group of toxins that pose a severe threat to human, animal, and/or plant health and they have been designated as Select Toxins. The Federal Select Agent Program oversees possession, use, and transfer of these toxins. Registration with the CDC or USDA is required for possession, use, modification, production, storage, and/or transfer of non-exempt quantities of Select Toxins. Exempt quantities that do not require registration still need to be carefully managed to prevent loss or misuse. For further information related to Select Toxins please use the following links:

• Select Agents and Toxins
• https://www.selectagents.gov/ 

The University is committed to the humane care and use of animals in research. The use of animals in research, teaching, field studies and outreach activities is subject to state and federal laws and guidelines. University policy specifies that:

• All animals under university care will be treated humanely and users of vertebrate animals must follow policies set forth in the Guide for the Care and Use of Laboratory Animals published by the Public Health Service, National Institutes of Health (NIH).
• Research or teaching activities involving the use of vertebrate animals are regulated by laws and policies at the campus, state and federal levels.
• All animal projects must receive approval by the Institutional Animal Care and Use Committee (IACUC) prior to conducting work.
• All individuals involved in activities in which animals are used must attend a training session on the humane care and use of animals conducted by the Director of the DLAR or designee as required in SUNY Stony Brook’s Assurance filed with the National Institutes of Health.

Project directors are responsible for the care and treatment of animals under their supervision, and for adherence to applicable University, state and federal regulations. Project directors planning to carry out research using vertebrate animals can visit the Office of Research Compliance (ORC) for further information and protocol submission to the IACUC for review.

Plant research is vital and can help to answer important questions related to agriculture, the environment, and basic life processes. In general, plant research does not generally pose a risk to human health. However, research involving plant diseases, genetically modified plants, or plant pests and organisms associated with plants can pose a hazard and be detrimental to agricultural crops and the environment. Therefore, specific levels of containment may be required to prevent any potential release of any modified plant or associated organisms. The movement, use, possession or release of exotic or potentially harmful plant-associated arthropods, biological control agents, plant pests, plant pathogens, noxious weeds and invasive plants are regulated by local, state, and federal agencies.

Regulation and Oversight
Transgenic plants and plant pests are subject to federal guidelines, regulations, and rules pertaining to their containment, movement, and release into the environment. In addition, States may have applicable regulations as well.

NIH Guidelines
The NIH Guidelines describes and details risk assessment and containment measures for various biological experiments involving the use of recombinant or synthetic nucleic acid molecules (rsNAM). These guidelines originally focused on experiments conducted with rsNAM microorganisms. However, the Guidelines have been expanded over the years to include a wide range of federally funded research projects, including plant research. Appendix L of the NIH guidelines provide information related to physical and biological containment (i.e. Plant Biosafety Levels BL1-P to BL4-P and growth chambers) for rsNAM research involving plants, plant-associated pests, animals, and plant pathogens in greenhouses. Any researcher conducting experiments with transgenic plants and/or plant associated organisms must receive IBC approval before beginning such work.

USDA-APHIS
The USDA Animal and Plant Health Inspection Service (APHIS) has authority under the Federal Plant Protection Act to protect U.S. agriculture from pests and disease. This includes rsNAM-containing plants and potential plant pests. There are two operational programs within APHIS devoted to plants and their associated organisms.

Plant Protection and Quarantine (PPQ)
Focuses on risks associated with entry, establishment, or spread of animal and plant pests. Permits are required for the importation into the U.S. and transit through the U.S. of regulated plants and plant products for consumption or propagation.

Biotechnology Regulatory Services (BRS)
Focuses on the risks associated with the introduction of organisms developed using genetic engineering that may pose a danger to U.S. agriculture and the environment. Permits are required for the importation, interstate movement, or environmental release of genetically engineered organisms that pose a risk to plant health.

More detailed information can be found at https://www.aphis.usda.gov/

HHS – USDA Select Agents and Toxins
Select Agents and Toxins (SAT) are biological agents that have been determined to have the potential to pose a significant threat to the health of humans, animals, plants, or animal and plant products. The SAT program is jointly administered by the CDC (HHS) and APHIS (USDA). APHIS is the lead agency for regulating agricultural pests and products. Lists of select agents and regulations can be found at https://www.selectagents.gov/.

Plant Biosafety Levels
Research involving plants or plant-associated pests, generally, does not pose a hazard to humans but that does not mean that there is no reason for concern. The main reason for biocontainment regarding plant research is to prevent the transfer of propagules and other organisms from a containment area to agricultural areas or the environment. Four levels of biocontainment are described in Appendix L of the NIH Guidelines for greenhouses and growth chambers. Biosafety Levels provide a description of a combination of administrative controls, work practices and procedures, equipment and facility features required to achieve a designed level of containment. Experiments may be assigned to one of four levels of biosafety containment (BL1-P, BL2-P, BL3-P, & BL4-P).

• Biosafety Level 1 for Plants (BL1-P): BL1-P is designed to provide a low level of containment for experiments involving transgenic plants where there is no evidence that a modified organism would be able to survive and spread in the environment and if accidentally released would not pose an environmental risk.
• Biosafety Level 2 for Plants (BL2-P): BL2-P is assigned to experiments with transgenic plants and associated organisms, which if released could be viable in the surrounding environment and could have a negligible impact or could be readily managed.
• Biosafety Level 3 & 4 for Plants (BL3-P & BL4-P): BL3-P & BL4-P containment is designed to prevent accidental release of transgenic plants and plant associated organisms and non-GE plant research that involves exotic infectious agents that have the potential for significant detrimental impact on agriculture and the environment. Stony Brook University does not currently have BL3-P or BL4-P containment facilities.

Plant Containment, Practices and Handling
Appropriate selection of the following biological containment practices may be used to meet containment requirements for a given organism. The present list is not exhaustive; there may be other ways of preventing effective dissemination that could possibly lead to establishment of the organism or it’s genetic material in the environment resulting in deleterious consequences to manage natural ecosystems.

Biological Containment Practices (Plants)
Effective dissemination of plants by pollen or seed can be prevented by one or more of the following procedures:

• Cover the reproductive structures to prevent pollen dissemination at flowering and seed dissemination at maturity.
• Remove reproductive structures by employing male sterile strains or harvest the plant material prior to the reproductive stage.
• Ensure that experimental plants flower at a time of year when cross-fertile plants are not flowering within the normal pollen dispersal range of the experimental plant.
• Ensure that cross-fertile plants are not growing within the known pollen dispersal range of the experimental plant.

Biological Containment Practices (Microorganisms)
Effective dissemination of microorganisms beyond the confines of the greenhouse can be prevented by one or more of the following procedures:

• Confine all operations to injections of microorganisms or other biological procedures (including genetic manipulation) that limit replication or reproduction of viruses and microorganisms or sequences derived from microorganisms and confine these injections to internal plant parts or adherent plant surfaces.
• Ensure that organisms which can serve as hosts or promote the transmission of the virus or microorganism, are not present within the farthest distance that the airborne virus or microorganism may be expected to be effectively disseminated.
• Conduct experiments at a time of year when plants that can serve as hosts are either not growing or are not susceptible to productive infection.
• Use viruses and other microorganisms or their genomes that have known arthropod or animal vectors, in the absence of such vectors.
• Use microorganisms that have an obligatory association with the plant.
• Use microorganisms that are genetically disabled to minimize survival outside of the research facility and whose natural mode of transmission requires injury of the target organism or assures that inadvertent release is unlikely to initiate productive infection of organisms outside of the experimental facility.

Biological Containment Practices (Macroorganisms)
Effective dissemination of arthropods and other small animals can be prevented by using one or more of the following procedures:

• Use non-flying, flight-impaired, or sterile arthropods.
• Use non-motile or sterile strains of small animals.
• Conduct experiments at a time of year that precludes the survival of escaping organisms.
• Use animals that have an obligatory association with a plant that is not present within the dispersal range of the organism.
• Prevent the escape of organisms present in run-off water by chemical treatment or evaporation of run-off water. Containment for arthropods is described in the Arthropod Containment Guidelines and Addendum 1 Containment Practices for Arthropods Modified with Engineered Transgenes Capable of Gene Drive.

Greenhouse Growth Chambers
Growth chambers are specially designed rooms, chambers or equipment that allow researchers to control the environmental conditions (i.e. humidity, light, temperature, etc.) when studying plants. Growth chambers can offer many advantages over the use of field plots and greenhouses and answer important questions about plant development and growth related to climate change and plant pests and pathogens. According to the NIH Guidelines growth chambers may be used for containment at BL1-P and BL2-P levels under certain conditions.

Field Work
Field work with transgenic plants requires permits from APHIS prior to the start of work. The permit process allows for the introduction (importation, interstate movement, or environmental release) of certain genetically engineered organisms under conditions determined and described in 7 CFR part 340. If you have any plans to do field work with genetically engineered plants or associated organisms, inquire about permits first.

Biohazardous Plant or Plant Associated Organism Waste
Experimental organisms and materials (i.e. plants, seeds, soil, fruit, etc.) utilizing BL1-P or BL2-P containment must be rendered biologically inactive by appropriate methods (i.e. autoclave or chemical inactivation, etc.) before disposal outside of the greenhouse facility. In addition, pots, trays, and other hard plastic horticultural supplies in shared use should be thoroughly cleaned and decontaminated to eliminate and prevent survival of plant material or pathogens.

Should you need further assistance or have questions or concerns regarding plant research conducted at Stony Brook University please contact Christopher Kuhlow, Biological Safety Officer at (631) 632-3717 or Christopher.Kuhlow@stonybrook.edu.