Institutional Biosafety Guidelines for Research Involving Recombinant DNA

Institutional Biosafety Committee Institutional Biosafety Guidelines for Research Involving Recombinant DNA
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Institutional Biosafety Committee Institutional Biosafety Guidelines for Research Involving Recombinant DNA

NYU’s Institutional Biosafety Committee guides researchers in registering experiments involving recombinant DNA, as well as in assessing the level of risk to an investigator, a study participant, or a laboratory worker.

Registering Recombinant DNA Experiments

If you plan to conduct experiments involving the use of recombinant DNA, your project must be registered with our Institutional Biosafety Committee for review and approval. Using your Kerberos ID, log on to Research Navigator and complete and submit the recombinant DNA experiment registration form. If the recombinant DNA experiment requires biosafety level (BL) 2 containment, the laboratory must be approved by Environmental Health and Safety.

Research involving human gene transfer must also be registered with and approved by our Institutional Biosafety Committee.

Determining the Biosafety Level of Research Involving Recombinant DNA

The level of risk to human health and the environment determines what approvals you need before an experiment begins.

In section III of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) six categories of experiments are described, as well as required approvals, risk levels, and levels of containment.

The three following components should be considered in determining the overall risk to human health or the environment:

  • The vector: for example, is it a plasmid, a pathogenic virus, or a plasmid that when transferred into a eukaryotic cell would produce a pathogenic virus?
  • The host: for example, is it a pathogen? See Appendix B of the NIH Guidelines, “Classification of Human Etiologic Agents on the Basis of Hazard.”
  • The DNA to be cloned: for example, does its expression confer pathogenicity on or increase the virulence of an otherwise nonpathogenic host? Does it change the range of a pathogenic host? Does it encode a toxin or a select agent?

For further guidance in determining the BL of recombinant DNA, see Section II-A of the NIH Guidelines.

Registrations involving human gene transfer are approved for one year. All other registrations are approved for three years. Researchers are required to submit an annual continuation to retain institutional approval and must submit an amendment whenever a change is proposed for any registration. Continuations and amendments allow the committee to determine whether risk has increased and a higher BL is indicated.

Determining a Substantial Change in Experimental Protocol

Usually, an increase in the risk level of a vector, host, or cloned DNA would require a new review by the Institutional Biosafety Committee.

Agents are classified by the NIH into four risk groups (RGs)—RG1, RG2, RG3, or RG4—according to their relative pathogenicity in healthy adult humans.

A change of vector to one in a higher RG that was not named in the original registration document would necessitate a review. For instance, if a principal investigator plans to use a vaccinia virus vector but did not indicate in the original document that a vector from RG2 would be used, a review would be indicated. Another instance in which a review would be required is a change of vector to one in a different taxonomic family, regardless of the BL.

A new host from a higher RG not included in the original registration document would also require review. For example, the original document indicated that Escherichia coli K-12 strains would be the only hosts, but now the principal investigator plans to use an RG2 bacterium such as Streptococcus pneumoniae as a host. Another example would be if cultured cells were the only eukaryotic host initially indicated, but the principal investigator now plans to use mice.

If a piece of DNA to be cloned confers pathogenicity or virulence in humans or animals and was not included in the original registration document, a review is needed. For example, the principal investigator plans to clone a DNA fragment in an E. coli host that confers on it the ability to colonize the human urinary tract.

The use of a new host or vector in the same RG as those indicated in the original registration document would not constitute a substantial change requiring review. Similarly, cloning DNA that differs from DNA proposed originally would not constitute a substantial change, as long as the DNA does not encode a human or animal toxin or confer or enhance pathogenicity in humans or animals.

Risk Assessment

Risk assessment is ultimately a subjective process. The investigator must make an initial risk assessment based on the agent’s RG. The four RGs are defined by the NIH.

Criteria for Risk Groups

Classification of agents in Appendix B of the NIH Guidelines, “Classification of Human Etiologic Agents on the Basis of Hazard,” is based on the potential effect of a biological agent on a healthy human adult and does not account for instances in which an individual may have increased susceptibility to such agents, such as preexisting diseases, medications, compromised immunity, or pregnancy or breastfeeding (which may increase exposure of infants to some agents). Personnel may need periodic medical surveillance to ascertain their fitness to perform certain activities; they may also need to be offered prophylactic vaccines and boosters (see IV-B-1-f in Section IV of the NIH Guidelines, “Responsibilities of the Institution, General Information”).

Comprehensive Risk Assessment

In deciding on the appropriate containment for an experiment, the first step is to assess the risk of the agent itself. Appendix B in the NIH Guidelines, “Classification of Human Etiologic Agents on the Basis of Hazard,” classifies agents into risk groups based on an assessment of their ability to cause disease in humans and the available treatments for such disease. Once the risk group of the agent is identified, this should be followed by a thorough consideration of how the agent is to be manipulated. 

Factors to be considered in determining the level of containment include agent factors such as the following:

  • virulence
  • pathogenicity
  • infectious dose
  • environmental stability
  • route of spread
  • communicability
  • operations to be performed
  • quantity of rDNA-containing organism being produced
  • availability of vaccine or treatment
  • gene product effects such as toxicity, physiological activity, and allergenicity

Any strain that is known to be more hazardous than the parent (wild-type) strain should be considered for handling at a higher containment level. Certain attenuated strains or strains that have been demonstrated to have irreversibly lost known virulence factors may qualify for a reduction in containment level, as compared to the RG assigned to the parent strain (see V-B in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”).

A final assessment of risk based on these considerations is then used to set the appropriate containment conditions for the experiment (see II-B in Section II of the NIH Guidelines, “Containment”). The containment level required may be equivalent to the RG classification of the agent, or it may be raised or lowered as a result of the above considerations.

The Institutional Biosafety Committee must approve the risk assessment and the biosafety containment level for recombinant DNA experiments described in Section III of the NIH Guidelines, “Experiments That Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation” (III-A); “Experiments That Require NIH OSP and Institutional Biosafety Committee Approval Before Initiation” (III-B); “Experiments That Require Institutional Biosafety Committee and Institutional Review Board Approvals and RAC Review (if Applicable) before Research Participant Enrollment” (III-C); and “Experiments That Require Institutional Biosafety Committee Approval before Initiation” (III-D).

Careful consideration should be given to the types of manipulation planned for some higher-RG agents. For example, the RG2 dengue viruses may be cultured under biosafety level (BL) 2 containment (see Section II-B of the NIH Guidelines); however, when such agents are used for animal inoculation or transmission studies, a higher containment level is recommended. Similarly, RG3 agents such as Venezuelan equine encephalomyelitis and yellow fever viruses should be handled at a higher containment level for animal inoculation and transmission experiments.

Researchers working with HIV, hepatitis B virus (HBV) or other blood-borne pathogens should consult the applicable Occupational Safety and Health Administration (OSHA) regulation, 29 CFR 1910.1030, and OSHA publication 3186. BL2 containment is recommended for all activities involving blood-contaminated clinical specimens, body fluids, and tissues from all humans, or laboratory animals infected or inoculated with HIV or HBV. 

Activities such as producing research laboratory–scale quantities of HIV or other blood-borne pathogens, manipulating concentrated virus preparations, or conducting procedures that may produce droplets or aerosols are performed in a BL2 facility using the additional practices and containment equipment recommended for BL3. 

Activities involving industrial-scale volumes or preparations of concentrated HIV are conducted in a BL3 facility, or BL3 large scale facility if appropriate, using BL3 practices and containment equipment.

Exotic plant pathogens and animal pathogens of domestic livestock and poultry are restricted and may require special laboratory design, operation, and containment features not addressed in Biosafety in Microbiological and Biomedical Laboratories (see V-C in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”). For information regarding the importation, possession, or use of these agents, see V-G and V-H in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV.”

Containment

Effective biological safety programs have been operative in a variety of laboratories for many years. Considerable information already exists about the design of physical containment facilities and selection of laboratory procedures applicable to organisms carrying recombinant DNA (see V-B in Section V of the NIH Guidelines, Footnotes and References of Sections I through IV).

The existing programs rely upon mechanisms that can be divided into two categories: (I) a set of standard practices that are generally used in microbiological laboratories and (II) special procedures, equipment, and laboratory installations that provide physical barriers that are applied in varying degrees according to the estimated biohazard.

Four BLs are described in Appendix G of the NIH Guidelines, “Physical Containment.” These BLs consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed and are based on the potential hazards imposed by the agents used and for the laboratory function and activity. BL4 provides the most stringent containment conditions; BL1, the least stringent. 

Experiments involving recombinant DNA lend themselves to a third containment mechanism, namely, the application of highly specific biological barriers. Natural barriers exist that limit either (I) the infectivity of a vector or vehicle (plasmid or virus) for specific hosts or (II) its dissemination and survival in the environment. Vectors, which provide the means for recombinant DNA or host cell replication, or both, can be genetically designed to decrease, by many orders of magnitude, the probability of dissemination of recombinant DNA outside the laboratory (see Appendix I of the NIH Guidelines, “Biological Containment”).

Since these three means of containment are complementary, different levels of containment can be established that apply various combinations of the physical and biological barriers along with a constant use of standard practices. Categories of containment are considered separately in order that such combinations can be conveniently expressed in the NIH Guidelines.

Physical containment conditions within laboratories, described in Appendix G of the NIH Guidelines, “Physical Containment,” may not always be appropriate for all organisms because of their physical size, the number of organisms needed for an experiment, or the particular growth requirements of the organism. Likewise, biological containment for microorganisms described in Appendix I of the NIH Guidelines, “Biological Containment,” may not be appropriate for all organisms, particularly higher eukaryotic organisms. 

However, the NIH Guidelines provide significant information about the design of research facilities and experimental procedures that are applicable to organisms containing recombinant DNA that is either integrated into the genome or into microorganisms associated with the higher organism as a symbiont or pathogen or in another relationship.

This information describes facilities for physical containment of organisms, either plant or animal, used in nontraditional laboratory settings and special practices for limiting or excluding the unwanted establishment, transfer of genetic information, and dissemination of organisms beyond the intended location, based on both physical and biological containment principles. Research conducted in accordance with these conditions effectively confines the organism.

For research involving plants, four BLs (BL1-P through BL4-P) are described in Appendix P NIH Guidelines, “Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants.”

BL1-P is designed to provide a moderate level of containment for experiments for which there is convincing biological evidence that precludes the possibility of survival, transfer, or dissemination of recombinant DNA into the environment, or in which there is no recognizable and predictable risk to the environment in the event of accidental release.

BL2-P is designed to provide a greater level of containment for experiments involving plants and certain associated organisms in which there is a recognized possibility of survival, transmission, or dissemination of recombinant DNA–containing organisms but the biological impact of such an inadvertent release is predictably minimal.

BL3-P and BL4-P describe additional containment conditions for research with plants and certain pathogens and other organisms that require special containment because of their recognized potential for significant detrimental impact on managed or natural ecosystems.

BL1-P relies upon accepted scientific practices for conducting research in most ordinary greenhouse or growth-chamber facilities and incorporates accepted procedures for good pest control and cultural practices. BL1-P facilities and procedures provide a modified and protected environment for the propagation of plants and microorganisms associated with the plants and a degree of containment that adequately minimizes the potential for release of biologically viable plants, plant parts, and microorganisms associated with them.

BL2-P and BL3-P rely upon accepted scientific practices for conducting research in greenhouses with organisms infecting or infesting plants in a manner that minimizes or prevents inadvertent contamination of plants within or surrounding the greenhouse. BL4-P describes facilities and practices known to provide containment of certain exotic plant pathogens.

For research involving animals that are of a size or have growth requirements that preclude the use of conventional primary containment systems used for small laboratory animals, four BLs (BL1-N through BL4-N) are described in Appendix Q of the NIH Guidelines, “Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals.”

BL1-N describes containment for animals that have been modified by stable introduction of recombinant DNA, or DNA derived therefrom, into the germ line (transgenic animals) and experiments involving viable recombinant DNA–modified microorganisms and is designed to eliminate the possibility of sexual transmission of the modified genome or transmission of recombinant DNA–derived viruses known to be transmitted from animal parent to offspring only by sexual reproduction. Procedures, practices, and facilities follow classical methods of avoiding genetic exchange between animals.

BL2-N describes containment that is used for transgenic animals associated with recombinant DNA–derived organisms and is designed to eliminate the possibility of vertical or horizontal transmission. Procedures, practices, and facilities follow classical methods of avoiding genetic exchange between animals or controlling arthropod transmission.

BL3-N and BL4-N describe higher levels of containment for research with certain transgenic animals involving agents that pose recognized hazard.

In constructing the NIH Guidelines, it was necessary to define boundary conditions for the different levels of physical and biological containment and for the classes of experiments to which they apply. These definitions do not take into account all existing and anticipated information on special procedures that will allow particular experiments to be conducted under different conditions than indicated here without affecting risk. Individual investigators and Institutional Biosafety Committees are urged to devise simple and more effective containment procedures and to submit recommended changes in the NIH Guidelines to permit the use of these procedures.

Risk Groups

The following list reflects the current state of knowledge and should be considered a resource document. Included are the more commonly encountered agents; the list is not meant to be all-inclusive. Information on agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH publication Biosafety in Microbiological and Biomedical Laboratories (see V-C, V-D, V-E, and V-F in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”). 

Further guidance on agents not listed here may be obtained through the Centers for Disease Control and Prevention, Biosafety Branch, Atlanta, GA 30333, Phone: 404-639-3883, Fax: 404-639-2294; the National Institutes of Health, Division of Safety, Bethesda, MD 20892, Phone: 301-496-1357; National Animal Disease Center, U.S. Department of Agriculture, Ames, IA 50010, Phone: 515-862-8258.

A special committee of the American Society for Microbiology will conduct an annual review of this appendix and its recommendation for changes will be presented to the Recombinant DNA Advisory Committee as proposed amendments to the NIH Guidelines.

Risk Group 1 Agents

RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include the following (see Section B-I in the NIH Guidelines): asporogenic Bacillus subtilis or Bacillus licheniformis; adeno-associated virus (AAV) types 1 through 4; and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and which are produced in the absence of a helper virus. A strain of Escherichia coli is an RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen) and (2) does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors.

Those agents not listed in RGs 2, 3, and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

Risk Group 2 Agents

Risk group 2 (RG2) agents are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available.

Risk Group 2—Bacterial Agents, Including Chlamydia

  • Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)
  • Actinobacillus
  • Actinomyces pyogenes (formerly Corynebacterium pyogenes)
  • Aeromonas hydrophila
  • Amycolata autotrophica
  • Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)
  • Arizona hinshawii—all serotypes
  • Bacillus anthracis
  • Bartonella henselae, B. quintana, B. vinsonii
  • Bordetella, including B. pertussis
  • Borrelia recurrentis, B. burgdorferi
  • Burkholderia (formerly Pseudomonas species), except those listed in B-III-A in Appendix B of the NIH Guidelines, which are in RG3)
  • Campylobacter coli, C. fetus, C. jejuni
  • Chlamydia psittaci, C. trachomatis, C. pneumoniae
  • Clostridium botulinum, Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicum, Cl. Tetani
  • Coxiella burnetii—specifically the phase II, Nine Mile strain, plaque purified, clone 4
  • Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale
  • Dermatophilus congolensis
  • Edwardsiella tarda
  • Erysipelothrix rhusiopathiae 
  • Escherichia coli—all enteropathogenic, enterotoxigenic, and enteroinvasive strains and strains bearing K1 antigen, including E. coli O157:H7
  • Francisella tularensis, specifically F. tularensis subspecies novicida (also known as F. novicida), strain Utah 112; F. tularensis subspecies holarctica LVS; F. tularensis biovar tularensis strain ATCC 6223 (also known as strain B38); (for research involving high concentrations, BL3 practices should be considered (see G-II-C-2 in Appendix G in the NIH Guidelines, “Special Practices (BL3)”).
  • Haemophilus ducreyi, H. influenzae
  • Helicobacter pylori
  • Klebsiella—all species except K. oxytoca, which is in RG1
  • Legionella, including L. pneumophila
  • Leptospira interrogans—all serotypes
  • Listeria
  • Moraxella
  • Mycobacterium (except those listed in B-III-A in Appendix B of the NIH Guidelines, including M. avium complex organisms, M. asiaticum, M. bovis bacilli Calmette Guérin (BCG) vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. avium paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi
  • Mycoplasma, except M. mycoides and M. agalactiae, which are restricted animal pathogens
  • Neisseria gonorrhoeae, N. meningitidis
  • Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis
  • Rhodococcus equi
  • Salmonella, including S. arizonae; S. choleraesuis; S. enteritidis; S. gallinarum-pullorum; S. meleagridis; S. paratyphi types A, B, and C; S. typhi, S. typhimurium
  • Shigella, including S. boydii, S. dysenteriae type 1, S. flexneri, S. sonnei
  • Sphaerophorus necrophorus
  • Staphylococcus aureus
  • Streptobacillus moniliformis
  • Streptococcus, including S. pneumoniae, S. pyogenes
  • Treponema pallidum, T. carateum
  • Vibrio cholerae, V. parahaemolyticus, V. vulnificus
  • Yersinia enterocolitica
  • Yersinia pestis, specifically strains lacking the 102 kb pigmentation locus and strains lacking the low calcium response plasmid

Risk Group 2—Fungal Agents

  • Blastomyces dermatitidis
  • Cladosporium bantianum, C. (Xylohypha) trichoides
  • Cryptococcus neoformans
  • Dactylaria galopava (Ochroconis gallopavum)
  • Epidermophyton
  • Exophiala (Wangiella) dermatitidis
  • Fonsecaea pedrosoi
  • Microsporum
  • Paracoccidioides brasiliensis
  • Penicillium marneffei
  • Sporothrix schenckii
  • Trichophyton

Risk Group 2—Parasitic Agents

  • Ancylostoma human hookworms, including A. duodenale, A. ceylanicum
  • Ascaris, including Ascaris lumbricoides suum
  • Babesia, including B. divergens, B. microti
  • Brugia filaria worms, including B. malayi, B. timori
  • Coccidia
  • Cryptosporidium, including C. parvum
  • Cysticercus cellulosae (hydatid cyst, larva of Taenia solium)
  • Echinococcus, including E. granulosis, E. multilocularis, E. vogeli
  • Entamoeba histolytica
  • Enterobius
  • Fasciola, including F. gigantica, F. hepatica
  • Giardia, including G. lamblia
  • Heterophyes
  • Hymenolepis, including H. diminuta, H. nana
  • Isospora
  • Leishmania, including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruviana, L. tropica
  • Loa loa filaria worms
  • Microsporidium
  • Naegleria fowleri
  • Necator human hookworms, including N. americanus
  • Onchocerca filaria worms, including, O. volvulus
  • Plasmodium, including simian species, P. cynomolgi, P. falciparum, P. malariae, P. ovale, P. vivax
  • Sarcocystis, including S. sui hominis
  • Schistosoma, including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi
  • Strongyloides, including S. stercoralis
  • Taenia solium
  • Toxocara, including T. canis
  • Toxoplasma, including T. gondii
  • Trichinella spiralis
  • Trypanosoma, including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi
  • Wuchereria bancrofti filaria worms

Risk Group 2—Viruses

Adenoviruses, Human—All Types

Alphaviruses (Togaviruses)—Group A Arboviruses

  • Chikungunya vaccine strain 181/25
  • Eastern equine encephalomyelitis virus
  • Venezuelan equine encephalomyelitis vaccine strains TC-83 and V3526
  • Western equine encephalomyelitis virus 

Arenaviruses

  • Junin virus candid #1 vaccine strain
  • Lymphocytic choriomeningitis virus (nonneurotropic strains)
  • Tacaribe virus complex
  • Other viruses as listed in the reference source (see V-C in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Bunyaviruses

  • Bunyamwera virus
  • Rift Valley fever virus vaccine strain MP-12
  • Other viruses as listed in the reference source (see V-C in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Caliciviruses

Coronaviruses

Flaviviruses—Group B Arboviruses

  • Dengue virus serotypes 1, 2, 3, and 4
  • Japanese encephalitis virus strain SA 14-14-2
  • Yellow fever virus vaccine strain 17D
  • Other viruses as listed in the reference source (see V-C in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Hepatitis A, B, C, D, and E Viruses

Herpesviruses—Except Herpesvirus simiae (Monkey B Virus) (see B-IV-D in Appendix B of the NIH Guidelines, “Risk Group 4 (RG4)—Viral Agents”)

  • Cytomegalovirus
  • Epstein–Barr virus
  • Herpes simplex types 1 and 2
  • Herpes zoster
  • Human herpesvirus types 6 and 7

Orthomyxoviruses

  • Influenza viruses types A, B, and C (except those listed in B-III-D in Appendix B of the NIH Guidelines, “Risk Group 3 (RG3)—Viruses and Prions”)
  • Tick-borne orthomyxoviruses

Papovaviruses

  • All human papillomaviruses

Paramyxoviruses

  • Newcastle disease virus
  • Measles virus
  • Mumps virus
  • Parainfluenza viruses types 1, 2, 3, and 4
  • Respiratory syncytial virus

Parvoviruses

  • Human parvovirus (B19)

Picornaviruses

  • Coxsackie viruses types A and B
  • Echoviruses—all types
  • Polioviruses—all types, wild and attenuated
  • Rhinoviruses—all types

Poxviruses—All Types Except Monkeypox Virus (See B-III-D in Appendix B of the NIH Guidelines, “Risk Group 3 (RG3)—Viruses and Prions”) and Restricted Poxviruses, Including Alastrim, Smallpox, and Whitepox (See V-L in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Reoviruses—All Types, Including Coltivirus, Human Rotavirus, and Orbivirus (Colorado Tick Fever Virus)

Rhabdoviruses

  • Rabies virus—all strains
  • Vesicular stomatitis virus nonexotic strains: VSV-Indiana 1 serotype strains (e.g. Glasgow, Mudd-Summers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g., Ogden, Hazelhurst) 

Rubivirus (Togaviruses)

  • Rubella virus

Risk Group 3 Agents

RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.

Risk Group 3—Bacterial Agents, Including Rickettsia

  • Bartonella
  • Brucella, including B. abortus, B. canis, B. suis
  • Burkholderia (Pseudomonas) mallei, B. pseudomallei
  • Coxiella burnetii (except the phase II, Nine Mile strain listed in B-II-A in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Bacterial Agents Including Chlamydia”)
  • Francisella tularensis (except those strains listed in B-II-A in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Bacterial Agents Including Chlamydia”)
  • Mycobacterium bovis (except BCG strain, see B-II-A in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Bacterial Agents Including Chlamydia”), M. tuberculosis
  • Orientia tsutsugamushi (formerly R. tsutsugamushi)
  • Pasteurella multocida type B—“buffalo” and other virulent strains
  • Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R. siberica, R. typhi (R. mooseri)
  • Yersinia pestis (except those strains listed in B-II-A in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Bacterial Agents Including Chlamydia”)

Risk Group 3—Fungal Agents

  • Coccidioides immitis (sporulating cultures; contaminated soil)
  • Histoplasma capsulatum, H. capsulatum var. duboisii

Risk Group 3—Parasitic Agents

None

Risk Group 3—Viruses and Prions

Alphaviruses (Togaviruses)—Group A Arboviruses

  • Chikungunya virus (except the vaccine strain 181/25 listed in B-II-D in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Viruses”)
  • Semliki Forest virus
  • St. Louis encephalitis virus
  • Venezuelan equine encephalomyelitis virus, except the vaccine strains TC-83 and V3526 (see B-II-D in Appendix B of the NIH Guidelines, “Risk Group 2 (RG2)—Viruses”)
  • Other viruses as listed in the reference source (see Section V-C of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Arenaviruses

  • Flexal
  • Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)

 Bunyaviruses 

  • Hantaviruses, including Hantaan virus
  • Rift Valley fever virus

Coronaviruses

  • Severe acute respiratory syndrome (SARS)–associated coronavirus (SARS-CoV)
  • Middle East respiratory syndrome coronavirus (MERS-CoV)

Flaviviruses (Togaviruses)—Group B Arboviruses

  • Japanese encephalitis virus (except those strains listed in B-II-D in Appendix B of the NIH Guidelines, “Risk Group2 (RG2)—Viruses”)
  • Yellow fever virus
  • Other viruses as listed in the reference source (see Section V-C of the NIH Guidelines, “Footnotes and References of Sections I through IV”)

Orthomyxoviruses

  • Influenza viruses 1918–1919 H1N1 (1918 H1N1), human H2N2 (1957–1968), and highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage

Poxviruses

  • Monkeypox virus

Prions

  • Transmissible spongiform encephalopathies (TSE) agents (Creutzfeldt–Jakob disease and kuru agents) (see V-C in Section V of the NIH Guidelines, “Footnotes and References of Sections I through IV” for containment instruction)

Retroviruses

  • HIV types 1 and 2
  • Human T cell lymphotropic virus types 1 and 2
  • Simian immunodeficiency virus

Rhabdoviruses

  • Vesicular stomatitis virus, except those strains listed in B-II-D in Appendix B of the NIH Guidelines, “Risk Group2 (RG2)—Viruses”

Risk Group 4 Agents

RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

Risk Group 4—Bacterial Agents

None

Risk Group 4—Fungal Agents

None

Risk Group 4—Parasitic Agents

None

Risk Group 4—Viral Agents

Arenaviruses

  • Guanarito virus
  • Lassa virus
  • Junin virus, except the candid #1 vaccine strain listed in B-II-D in Appendix B of the NIH Guidelines, “Risk Group2 (RG2)—Viruses”
  • Machupo virus
  • Sabia

Bunyaviruses (Nairovirus)

  • Crimean–Congo hemorrhagic fever virus

Filoviruses

  • Ebola virus
  • Marburg virus

Flaviviruses (Togaviruses)—Group B Arboviruses

  • Tick-borne encephalitis virus complex, including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring–summer encephalitis viruses

Herpesviruses (alpha)

  • Herpesvirus simiae (Herpes B or Monkey B virus)

Paramyxoviruses

  • Equine morbillivirus (Hendra virus)

Hemorrhagic fever agents and viruses as yet undefined

Animal Viral Etiologic Agents in Common Use

The following list of animal etiologic agents is appended to the list of human etiologic agents. None of these agents is associated with disease in healthy adult humans; they are commonly used in laboratory experimental work.

A containment level appropriate for RG1 human agents is recommended for their use. For agents that are infectious to human cells, such as amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.

Baculoviruses

Herpesviruses

  • Herpesvirus ateles
  • Herpesvirus saimiri
  • Marek’s disease virus
  • Murine cytomegalovirus

Papillomaviruses

  • Bovine papilloma virus
  • Shope papilloma virus

Polyoma viruses

  • Polyoma virus
  • Simian virus 40 (SV40) 

Retroviruses

  • Avian leukosis virus
  • Avian sarcoma virus
  • Bovine leukemia virus
  • Feline leukemia virus
  • Feline sarcoma virus
  • Gibbon leukemia virus
  • Mason–Pfizer monkey virus
  • Mouse mammary tumor virus
  • Murine leukemia virus
  • Murine sarcoma virus
  • Rat leukemia virus

Murine Retroviral Vectors

Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50 percent of their respective parental viral genome and that have been demonstrated to be free of detectable replication-competent retrovirus can be maintained, handled, and administered under BL1 containment.