4. Class III-D experiments

Class III-D experiments require IBC approval and submission of an rDNA Registration Document prior to initiation. These experiments involve:

  1. The use of Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as host-vector systems
  2. Cloning DNA from Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents
  3. The use of infectious DNA or RNA viruses or defective DNA or RNA viruses
  4. Whole animals
  5. Whole plants
  6. More than 10 liters of culture
  7. Experiments Involving Influenza Virus

The use of Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as host-vector systems.

  • Experiments involving the introduction of rsNAM into Risk Group 2 agents will usually be conducted at Biosafety Level (BSL) 2 containment. Experiments with such agents will usually be conducted with whole animals at BSL2 or Animal BSL (ABSL) 2 containment.
  • Experiments involving the introduction of rsNAM into Risk Group 3 agents will usually be conducted at BSL3 containment. Experiments with such agents will usually be conducted with whole animals at BSL3 or ABSL3 containment.
  • Experiments involving the introduction of rsNAM into Risk Group 4 agents shall be conducted at BSL4 containment. Experiments with such agents will usually be conducted with whole animals at BSL4 or ABSL4 containment; however, no BSL4 facility is available for such work at the University of Iowa.
  • Containment conditions for experiments involving the introduction of rsNAM into restricted agents shall be set on a case-by-case basis following NIH/OBA review. A USDA/APHIS permit is required for work with plant or animal pathogens. Experiments with such agents shall be conducted with whole animals at BSL4 or ABSL4 containment; however, as noted above, no BSL4 facility is available for working with animals requiring ABSL4 containment.

Cloning DNA from Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents into nonpathogenic prokaryotic or lower eukaryotic host-vector systems.

  • Experiments in which DNA from Risk Group 2 or 3 agents is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BSL2 containment. Experiments in which DNA from Risk Group 4 agents is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BSL2 containment after demonstration that only a totally and irreversibly defective fraction of the agent’s genome is present in a given recombinant. In the absence of such a demonstration, BSL4 containment shall be used. The IBC may approve the specific lowering of containment for particular experiments to BSL1. Many experiments in this category are exempt from the NIH Guidelines (see Section III-F); however, note that the IBC requires that all research involving recombinant or synthetic nucleic acids be registered with the IBC (see VII). Experiments involving the formation of rsNAM for certain genes coding for molecules toxic for vertebrates require NIH/OBA approval (see Section III-B-1) or shall be conducted under NIH specified conditions as described in Appendix F of the NIH Guidelines.
  • Containment conditions for experiments in which DNA for restricted agents is transferred into nonpathogenic prokaryotes or lower eukaryotes shall be determined by NIH/OBA following a case-by-case review. A USDA permit is required for work with plant or animal pathogens.

The use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems.

  • Experiments involving the use of infectious or defective Risk Group 2 viruses in the presence of helper virus may be conducted at BSL2.
  • Experiments involving the use of infectious or defective Risk Group 3 viruses in the presence of helper virus may be conducted at BSL3.
  • Experiments involving the use of infectious or defective Risk Group 4 viruses in the presence of helper virus may be conducted at BSL4.
  • Experiments involving the use of infectious or defective restricted poxviruses in the presence of helper virus shall be determined on a case-by-case basis following NIH/OBA review.
  • Experiments involving the use of infectious or defective viruses in the presence of helper virus which are not covered in Sections III-D-3-a through III-D-3-d may be conducted at BSL1.

Whole animals.

  • rsNAM, or DNA or RNA molecules derived therefrom, from any source except for greater than two-thirds of eukaryotic viral genome may be transferred to any non-human vertebrate or any invertebrate organism and propagated under conditions of physical containment comparable to BSL1 or ABSL1 and appropriate to the organism under study.
  • Animals that contain sequences from viral vectors, which do not lead to transmissible infection either directly or indirectly as a result of complementation or recombination in animals, may be propagated under conditions of physical containment comparable to BSL1 or ABSL1 and appropriate to the organism under study.
  • For experiments involving rsNAM, or DNA or RNA derived therefrom, involving whole animals, including transgenic animals, and not covered by Sections III-D-1 or III-D-4-a, the appropriate containment shall be determined by the IBC.
  • Exceptions under Section III-D-4, Experiments Involving Whole Animals:
    • Experiments involving the generation of transgenic rodents that require BSL1 containment are described under Section III-E-3, Experiments Involving Transgenic Rodents.
    • The purchase or transfer of transgenic rodents is exempt from the NIH Guidelines under Section III-F, Exempt Experiments (see Appendix C-VI, The Purchase or Transfer of Transgenic Rodents).

Whole plants.

  • Plant BSL (BSL-P) 3 or BSL2-P+ biological containment is recommended for experiments involving most exotic infectious agents with recognized potential for serious detrimental impact on managed or natural ecosystems when rsNAM techniques are associated with whole plants.
  • BSL3-P or BSL2-P+ biological containment is recommended for experiments involving plants containing cloned genomes of readily transmissible exotic infectious agents with recognized potential for serious detrimental effects on managed or natural ecosystems in which there exists the possibility of reconstituting the complete and functional genome of the infectious agent by genomic complementation in planta.
  • BSL4-P containment is recommended for experiments with a small number of readily transmissible exotic infectious agents, such as the soybean rust fungus (Phakospora pachyrhizi) and maize streak or other viruses in the presence of their specific arthropod vectors that have the potential of being serious pathogens of major U.S. crops.
  • BSL3-P containment is recommended for experiments involving sequences encoding potent vertebrate toxins introduced into plants or associated organisms. rsNAM containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD50 of <100 nanograms per kilogram body weight fall under Section III-B-1 and require NIH/OBA and IBC approval before initiation.
  • BSL3-P or BSL2-P+ biological containment is recommended for experiments with microbial pathogens of insects or small animals associated with plants if the rsNAM modified-organism has a recognized potential for serious detrimental impact on managed or natural ecosystems.

More than 10 liters of culture.

  • The appropriate containment will be decided by the IBC. Where appropriate, Appendix K. Physical Containment for Large Scale Uses of Organisms Containing Recombinant or Synthetic Nucleic Acid Molecules, shall be used.

Experiments Involving Influenza Viruses

  • Experiments with influenza viruses containing the H2 hemagglutinin (HA) segment shall be conducted at BSL3 enhanced. Experiments with the H2 HA gene in cold-adapted, live attenuated vaccine strains may be conducted at BSL2 containment provided segments with mutations conferring temperature sensitivity and attenuation are not altered in the recombinant or synthetic virus. Experiments with risk Group 2 influenza viruses containing genes from human H5N1 other than the HA gene can be worked on at BSL2.
  • Experiments involving influenza viruses containing a majority of genes and/or segments from a HPAI H5N1 influenza virus shall be conducted at BSL3 enhanced containment. Experiments involving influenza viruses containing a minority of genes and/or segments from a HPAI H5N1 influenza virus shall be conducted at BSL3 enhanced unless a risk assessment performed by the IBC determines that they can be conducted safely at BSL2 and after they have been excluded pursuant to 9 CFR 121.3(e).
  • Experiments involving influenza viruses containing any gene or segment from 1918 H1N1 shall be conducted at BSL3 enhanced containment.
  • The availability of antiviral drugs as preventive and therapeutic measures is an important safeguard for experiments with 1918 H1N1, HPAI, H5N1, and human H2N2 (1957-1968). If an influenza virus containing genes from one of these viruses is resistant to both classes of current antiviral agents, adamantanes and neuraminidase inhibitors, higher containment may be required based on the risk assessment considering transmissibility to humans, virulence, pandemic potential, alternative antiviral agents if available, etc.