RECOMBINANT DNA REGISTRATION FORM INSTRUCTIONS
Any use of microbiological/infectious agents or recombinant DNA molecules in research must be reviewed and approved or
exempt by the Institutional Biosafety Committee before the procedure is initiated. The NIH “Guidelines for Research
Involving Recombinant DNA Molecules” contains requirements for protocol review, safe practices and necessary equipment.
Familiarity with the appropriate NIH guideline is required when determining if an application is required and for laboratory
operation with either exempt or approved protocols. Renewal is required every two years.
PI information fields are self-explanatory. Make sure that ALL fields are filled.
RESEARCH DESCRIPTION
Describe your research project in terms of rDNA. Indicate what rDNA molecules will be created or procured from other
labs/collections/commercially. Explain WHAT you are going to do with rDNA. Please remember that IBC is not a granting
agency, it is concerned with safety of your rDNA research, not with your science.
Please DO write:
We will anneal synthetic oligonucleotides and clone them in a retroviral shRNA expression vector (name and source of the
vector). The resulting construct will be used to generate retroviral supernatants by means of co-transfection with VSVG-coat
protein encoding plasmid (name of the plasmid) into a packaging cell line Phoenix gp. These supernatants will be
subsequently used to infect our favorite cell line (name of the cell line). Infected cells will be selected for antibiotic
resistance, tested for mycoplasma contamination, and injected into our favorite experimental animal (animal species).
Please do NOT write:
Viral-based gene silencing occupies a unique niche as a tool for delivering genetic information into a cell. In cases where the
cell line of interest is refractory to transfection (e.g., by lipid-mediated methods) or when long-term gene silencing is needed
to assess experimental outcome, delivery of silencing reagents using viral vectors serves as the method of choice. To that
end we wish to utilize a platform utilizing the lentivirus to deliver short hairpin RNA (shRNA) silencing constructs, each of
which has its own set of advantages. We will utilize a commercially available system that utilizes proprietary state-of-the-artcutting-edge-super-duper platform in order to exploit well-characterized properties associated with stability and broad
tropism as well as the high titers that can be achieved.
PROVIDE A NON-TECHNICAL SUMMARY OF rDNA USE FOR THIS REGISTRATION.
The NIH mandates for IBC to have a broad representation, including community members. Some IBC members may not be
as familiar with your area of research as you are. BE INFORMATIVE. Provide RELEVANT (as far as rDNA and biosafety
go) information only. Try to use plain language and avoid technical terms and jargon. Failure to do so may result in delays
with your protocol approval.
Please DO write:
We will use rDNA methodology to construct a defective retrovirus. The defect makes this retrovirus unable to replicate under
most circumstances. The introduction of this recombinant retrovirus into our favorite cells (name of the cell line) will result (is
expected to result) in the reduction of synthesis of our favorite protein (such-and-such). We will introduce recombinant
retrovirus in our favorite cells, and then inject these modified cells in our favorite experimental animals (name animals) to
study very important biological problem (such-and-such).
Please do NOT write:
Our lab is interested in the molecular and genetic events that are involved in the progression of very important disease. We
have found our favorite protein to be highly up-regulated in gene expression in this disease. We wish to further examine the
role of this gene as it relates to disease, hypothesizing that our favorite protein over-expression is involved with a disease
phenotype. In vitro assays revealed a significant correlation of our favorite protein activity with the disease. In the proposed
in vivo study, we wish to utilize a commercially available technique of RNA interference (RNAi) as a way of “knocking down"
the gene of interest. Gene knockdown by RNAi can be mediated by synthetic molecules (e.g., chemically synthesized
siRNAs) or induced by short hairpin RNA (shRNA) expression constructs introduced into cells by transfection (e.g., with
plasmids) or viral transduction.
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The above passage in informative, but most information is general, and provides little insight into how rDNA will be used in
the proposed studies. The IBC will definitely require clarifications.
Check the appropriate registration category for experiments covered by NIH Guidelines:
This section is fairly self-explanatory, but may require the PI refer to the NIH guidelines themselves. The full text of
Guidelines can be found on IBC’s webpage or here http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html. The most
common difficulty associated with choosing the category is outdated wording found in NIH rDNA Guidelines in section D.III.
It reads:
Experiments involving the use of infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence
of helper virus in tissue culture systems.
The generation of replication-deficient retro- lenti- or adenoviral vectors does not appear to explicitly fall in this category
because of these vectors are infectious (in the sense that they are unable to cause an infection), nor the presence of helper
virus is not required in most cases. However, for NIH this category should be selected (see e.g.
http://www4.od.nih.gov/oba/RAC/Guidance/LentiVirus_Containment/index.htm). Therefore, please check D.III category for
projects that propose generation of retro-, lenti- or adenovirus vectors.
Please choose one category only (i.e. A, B, C, D, E, or F). (justify if more than one category is checked)
1.
RECOMBINANT INSERT:
a. Source(s) of DNA/RNA sequences
Include:
1) genus, such as Homo, Mus, Rattus, etc.;
2) species, such as Homo sapiens, Mus musculus, etc.;
3) strain ( if applicable)
4) Entrez NCBI Accession number.
b. Nature of the insert or protein expressed, any oncogenic potential, the gene function, and any expected toxicity
of the gene products: (Attach a vector map or manufacturer’s product description)
Will transcription regulators be used? YES
NO
if yes, please list the transcription regulators used and if virulence will be affected:
2.
VECTOR
a. Host organism strain/cell lines for propagation of the recombinant:
name of the strain/cell line, ATCC number
b. and c. original form does not need to be changed
SECTION 3: GENE EXPRESSION, FUNCTION AND SOURCE
This section is self-explanatory. Please remember that shRNA is a product of the gene. Therefore, choose YES in
response to a question “Will you be expressing gene products from recombinant DNA”. In fact, it is only in very special
circumstances that no gene products will be expressed from rDNA
SECTION 4: FOR ANIMAL EXPERIMENTS ONLY
4a. Check appropriate box to indicate IACUC approval status. State “pending” for “approval date” if the IACUC protocol has
been submitted and is not yet approved.
4b. State the host species and describe the animal model. Discuss any possible clinical signs in the animal.
4c. If replication competence is anticipated (or even possible), please discuss.
4d. If there are any potential risks to personnel (from host shedding of the virus), please discuss.
4e. Discuss the containment facilities and the safeguards against transmission to personnel.
SECTION 5: SELECT AGENT AND TOXIN
See page 6 to check if any agents in use are on Select Agent List. If so, fill the table provided. If not, go to SECTION 6.
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SECTION 6: BIOSAFETY AND CONTAINMENT LEVELS:
6a: Containment levels requested:
The CDC-NIH has designated four different biosafety levels, BSL-l to BSL-4. Microorganisms are categorized into a
biosafety level. In addition, each biosafety level requires a different combination of laboratory practices and techniques,
safety equipment, and laboratory facilities as recommended by the Centers for Disease Control and the National Institutes of
Health in the publication "Biosafety in Microbiological and Biomedical Laboratories" (BMBL). These combinations of
equipment and work practices are designed to minimize the risk of infectious disease when working with microorganisms.
Definitions and information on organism classification and proper handling procedures can be found in this publication.
Biosafety checklists have been developed for BSL-l - BSL-3 laboratories and provided in the Biosafety Manual and
Exposure Control Plan as an attachment:
http://www.southalabama.edu/com/research/pdf/biomanual2007.doc
Examples of BSL categories: Risk assessment is ultimately a subjective process. The investigator must make an initial
risk assessment based on the Risk Group (RG) of an agent (see NIH GUIDELINES FOR RESEARCH INVOLVING
RECOMBINANT
DNA MOLECULES Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard). This appendix includes
those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if
inoculated into humans. Included are lists of representative genera and species known to be pathogenic; mutated,
recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are
excluded.
Check box below if applicable:
DNA introduced into experimental animals that fall under NIH Guidelines
Section III-D-4 of the NIH Guidelines for use of Recombinant DNA materials covers experiments involving whole animals in
which the animal's genome has been altered by stable introduction of rDNA or DNA derived there from, into the germ-line
(transgenic animals) and experiments involving viable rDNA modified microorganisms tested on the whole animal. For
additional information of these guidelines, please reference Section III-D-4 located on NIH website at:
http://www4.od.nih.gov/oba/rac/guidelines/guidelines.htm1
Gene therapy in humans:
Investigators who wish to introduce pathogens or recombinant genetic material into human subjects must receive
IBC approval in addition to IRB approval.
The IBC evaluates the protocol for patient, staff, and environmental safety issues in their review. The following documents
are required by the IBC for review of a Gene Transfer protocol:
1. Human Gene Transfer Supplement Form
2. Investigator's brochure (same one submitted to the IRB)
3. IRB Consent Form
4. Sponsor's Protocol
5. Appendix M, if applicable. Please note that many studies include an Appendix M written by the sponsoring company for a
PI at some other institution. The answers provided (who is doing the work, where will it be done, how many patients, etc.)
may or may not reflect
what will be done here at USA. In these case, the IBC requests that the investigator provide answers to the Appendix M
questions that specifically address the USA component(s) of the project.
6. Any NIH Recombinant Advisory Committee (RAC) communications/approvals
7. Any FDA communications/approvals
Other, please explain: (no further instructions necessary)
SECTION 7: PERSONNEL AND TRAINING
Self explanatory no needed for further instructions.
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(revised 7/2008)
IBC No.:
Date received:
THE UNIVERSITY OF SOUTH ALABAMA COLLEGE OF MEDICINE
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC)
Registration for Recombinant DNA Research
INSTRUCTIONS: To register recombinant DNA research with the Institutional Biosafety Committee (IBC), complete this
form as required using the most current “NIH Guidelines for Research Involving Recombinant DNA Molecules.” Submit a
registration form for each project involving recombinant DNA. Principal Investigator’s are obligated to have reviewed the
latest version of the NIH Guidelines in preparation for conducting rDNA research and submitting this application. The NIH
Guidelines can be accessed at http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html
Return the completed form to the Office of Research Compliance, CSAB 128, Attn: Dusty Layton. Incomplete forms will be
returned.
Principal Investigator:
Department:
Mailing Address:
Bldg/Laboratory Room(s): (location of work):
Project Title:
Telephone:
FAX:
Email:
Research Description: (specifically address the use of rDNA and describe work in the lab directly
related to biological hazard agents)
Provide a non-technical summary of recombinant DNA use for this registration
(This should be written so that someone who is not an expert in your particular field can understand your
recombinant DNA use)
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Check the appropriate registration category for experiments covered by NIH Guidelines:
A.
Experiments that require IBC approval, Research Advisory Committee (RAC) review, and NIH Director
Approval before initiation.
Major actions under the NIH guidelines cannot be initiated without submission of relevant information on the proposed experiment
to the Office of Biotechnology Activities (OBA), NIH. (See Section III-A-1 of the NIH Guidelines)
Deliberate transfer of drug resistance trait to microorganisms that are unknown to acquire the trait naturally (see Section
V-B, Footnotes and References of Sections I-IV) if such acquisition could compromise the use of the drug to control
disease agents in humans, veterinary medicine, agriculture, will be reviewed by RAC.
B.
Experiments that require NIH/OBA and Institutional Biosafety Committee Approval before initiation.
Experiments involving the cloning of toxin molecules with LD50 of less than 100 nonograms per kilogram body weight.
C.
Experiments that require Institutional Biosafety Committee and Institutional Review Board Approvals plus
RAC review before participant enrollment.
Experiments involving the deliberate transfer of recombinant DNA, or DNA or RNA derived from recombinant DNA, into
one or more human research participants (human gene transfer).
D.
Experiments that require only Institutional Biosafety Committee Approval before initiation.
Experiments using Risk Group 2, 3, 4 or restricted agents as Host-Vector systems
Experiments in which DNA from Risk Group 2, 3, 4 or restricted agents is cloned into nonpathogenic prokaryotic or lower
eukaryotic host-vector systems
Experiments involving 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 whole animals
Experiments involving whole plants
Experiments involving more than 10 liters of culture
E.
Experiments that require Institutional Biosafety Committee notice simultaneous with initiation.
Experiments involving the formation of recombinant DNA molecules containing no more than two-thirds of the genome of
any eukaryotic virus
Experiments involving whole plants
Experiments involving transgenic rodents
F.
Exempt Experiments (Please complete the entire form for IBC review) (See Guidelines Section III-F 1 through 6). If
your research falls under one of the following, select the appropriate category of experiments:
Those that are not in organisms or viruses;
Those that consist entirely of DNA segments from a single non-chromosomal or viral DNA source, though one or more of
the segments may be a synthetic equivalent;
Those that consist entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated
only in that host (or a closely related strain of the same species), or when transferred to another host by well established
physiological means;
Those that consist entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids (but
excluding viruses) when propagated only in the host (or a closely related strain of the same species);
Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes,
though one or more of the segments may be a synthetic equivalent. See Appendices A-I through A-VI, Exemptions Under
Section III-F-5 – Sublists of Natural Exchangers, for a list of natural exchangers that are exempt from the NIH Guidelines;
or
Those that do not present a significant risk to health or the environment (see section IV-C-1-b-(1)-(c), Major Actions)
1.
RECOMBINANT INSERT:
(Add page(s) if needed)
2.
a.
Source(s) of DNA/RNA sequences (include genus, species, or microbiological agents):
b.
What is the biological activity of the gene product or sequence inserted?
VECTOR:
a.
Host organism strain/cell lines for propagation of the recombinant:
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b.
Vectors to be used (plasmids, phages, viruses) - specify type and strain, and give description. (Provide
commercial product literature, a web link to specific information, a vector map or a hard copy of any journals
describing construction of the vector).
c.
Is the vector replication competent?
YES
NO
If no, how is production of replication competent virus minimized?
3.
GENE EXPRESSION, FUNCTION & SOURCE:
a.
Will you be expressing gene products from recombinant DNA?
YES
NO
If yes, what are the gene product effects? (Specifically, it’s toxicity, physiological activity, allergenicity, oncogenic
potential or ability to alter cell cycle).
b.
Are any of the gene products potentially oncogenic or toxic vertebrates?
YES
NO
Discuss alternatives:
c.
Would any of the gene products increase the virulence of the recombinant virus?
YES
NO
Discuss:
d.
Will any gene be intentionally mutated?
YES
NO
Discuss:
4.
FOR ANIMAL EXPERIMENTS ONLY:
a.
b.
c.
Has IACUC approval been obtained?
YES
NO
If yes: IACUC protocol #:
IACUC approval date:
For in vivo vector administration discuss host species and target organs or systems.
Are helper viruses present in the host, which may lead to replication-competency for the recombinant
construct?
YES
NO
Discuss:
d.
e.
Are there biohazard implications including potential exposure to staff and animal colonies
YES
NO
Discuss. (Specifically address the potential for shedding of the virus in vivo from the animal host).
Describe the containment facilities where these experiments will take place.
SELECT AGENT AND TOXIN (CFR 42 Part 73):
5.
The USA Patriot Act of 2001 and the Public Health and Bioterrorism Preparedness Act of 2002 were signed into law by the
President on June 12, 2002. In order to comply with federal law, investigators must register the possession of Select Agents
with the IBC.


Are any of these agents restricted according to the enclosed Select Agent List? (see page 6)
If no, please skip to next section. If yes, please complete the Select Agent table below.
AGENT
Amount in Lab
Storage
Planned Use
Yes
No
Supplier
6
On the basis of this information you may be required to register your laboratory with the CDC Select Agent Program. The IBC will determine
if the agent/toxin meets exempt status and are exempt from registration with the CDC under 42 CFR 72.6 (h).
6.
BIOSAFETY AND CONTAINMENT LEVELS:
a.
Containment level requested:
BSL-1
BSL-2
Check box below if applicable:
BSL-3
BSL 1-3 Large Scale work (> than 10 liters)
DNA introduced into experimental animals that fall under NIH Guidelines:
Gene therapy in humans – Complete Human Gene Transfer Supplement Form
Other, please explain :
b.
Describe procedures for responding to an accidental spill and/or release :
c.
Identify potential exposure hazards during sample preparation and experimental manipulations (e.g.,
aerosol generation when transferring, mixing or centrifuging, use of sharps, waste disposal considerations, etc.)
d.
7.
Indicate any precautionary medical practices or advice :
PERSONNEL AND TRAINING:
List the names of all personnel involved in biohazardous research. (Add pages if needed)
Name
Title/Position
Biosafety Training
Current Status
___________________________________
_________________________________
Yes
No
___________________________________
_________________________________
Yes
No
___________________________________
_________________________________
Yes
No
___________________________________
_________________________________
Yes
No
___________________________________
_________________________________
Yes
No
NOTE: All personnel involved in biohazardous research must complete biosafety training before conducting
biohazardous procedures. The BBP training handout materials and post test is available on the biosafety
website at: http://www.southalabama.edu/com/research/biosafety.shtml. The post test and Hepatitis B
vaccination form should be submitted to the Office of Research Compliance
SIGNATURES AND ACKNOWLEDGMENT:
I acknowledge my responsibility as principal investigator for conducting this research in compliance with the
latest NIH recombinant DNA guidelines.
Signature of Investigator: _______________________________________________
Date: _____________________
Signature of Department Chair: _________________________________________
Date: ____________________
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APHIS Select Plant Pathogens, HHS Select Infectious Agents, and USDA Select Livestock Pathogens or Toxins
Updated: March 18, 2005
Viruses
1. African horse sickness virus 3
2. African swine fever virus 3
3. Akabane virus 3
4. Avian influenza virus (highly pathogenic) 3
5. Blue tongue virus (exotic) 3
6. Camel pox virus 3
7. Cercopithecine herpes 1 virus (Herpes B virus) 2
8. Classical swine fever virus 3
9. Crimean-Congo haemorrhagic fever virus 2
10. Eastern equine encephalitis virus 4
11. Ebola viruses 2
12. Foot and mouth disease virus 3
13. Goat pox virus 2
14. Japanese encephalitis virus 3
15. Lassa fever virus 2
16. Lumpy skin disease virus 3
17. Malignant catarrhal fever (Alcelaphine herpes virus
type 1)3
18. Marburg virus 2
19. Menangle virus 3
20. Monkeypox virus 2
21. Newcastle disease virus (velogenic) 3
-Avulavirus avian paramyxovirus 1 (highly virulent
strains)
22. Nipah and Hendra complex viruses 4
23. Peste des petits ruminants 3
24. Rift Valley fever virus 4
25. Rinderpest virus 3
26. Sheep pox virus3
27. South American haemorrhagic fever viruses [(Junin,
Machupo, Sabia, Flexal, Guanarito)] 2
28. Swine vesicular disease virus 3
29. Tick-borne encephalitis complex (flavi) viruses
[Central European Tick-borne encephalitis, Far
Eastern Tick-borne encephalitis (Russian Spring
and Summer encephalitis, Kyasanur Forest disease,
Omsk Hemorrhagic Fever)] 2
30. Variola major virus (Smallpox virus) and Variola
minor (Alastrim) 2
31. Venezuelan equine encephalitis virus 4
32. Vesicular stomatitis virus (exotic) 3
Prions
1. Bovine spongiform encephalopathy agent 3
1
APHIS Plant Pathogen
2
Toxins
1. Abrin (=> 100 mg) 2
2. Botulinum neurotoxins (=> 0.5 mg) 4
3. Clostridium perfringens epsilon toxin (=> 100 mg) 4
4. Conotoxins (=> 100 mg)2
5. Diacetoxyscirpenol (=> 1,000 mg) 2
6. Ricin (=> 100 mg) 2
7. Saxitoxin (=> 100 mg) 2
8. Shigatoxin (=>100 mg) 4
9. Shiga-like ribosome inactivating proteins (=> 100 mg) 4
10. Staphylococcal enterotoxins (=> 5 mg) 4
11. Tetrodotoxin ((=> 100 mg) 2
12. T– 2 toxin (=> 1,000 mg) 4
Exemptions
Bacteria
1. Bacillus anthracis 4
2. Botulinum neurotoxin producing strains of Clostridium 4
3. Brucella abortus 4
4. Brucella melitensis 4
5. Brucella suis 4
6. Burkholderia mallei 4
7. Burkholderia pseudomallei 4
8. Cowdria Ruminantium (Heartwater) 1
9. Coxiella burnetii 4
10. Francisella tularensis 3
11. Liberobacter africanus 1
12. Liberobacter asiaticus 1
13. Mycoplasma capricolum/M. F38/M. mycoides capri
(contagious caprine pleuropneumonia agent) 3
14. Mycoplasma mycoides mycoides (contagious bovine
pleuropneumonia agent) 3
15. Ralstonia solanacearum Race 3, biovar 2 1
16. Rickettsia prowazekii 2
17. Rickettsia rickettsii 2
18. Xanthomonas oryzae pv. oryzicola 1
19. Xylella fastidiosa (citrus variegated chlorosis strain) 1
20. Yersinia pestis 2
The following attenuated strains are exempt if used in basic or
applied research, as positive controls, for diagnostic assay
development, or the development of vaccines and therapeutics:
Fungi
1. Coccidioides immitis 4
2. Coccidioides posadasii 2
3. Peronosclerospora philippinensis 1
4. Sclerophthora rayssiae var zeae 1
5. Synchytrium endobioticum 1
HHS Select Infectious Agent
3
 The agents or toxins that are marked with amounts are
exempt if the aggregate amount under the control of a
principal investigator does not, at any time, exceed the
amount given.
The following agents or toxins are also exempt:
 Any agent or toxin that is in its naturally occurring
environment provided it has not been intentionally
introduced, cultivated, collected, or otherwise extracted from
its natural source (exclusion does not apply to plant
pathogens listed within Title 7 CFR Part 331 or Botulinum
neurotoxin producing strains of Clostridium)
 Non-viable select agent organisms or nonfunctional toxins.
Coccidioides posadasii ∆chs5 strain. Conotoxins specifically
excluded are: the class of sodium channel antagonist μconotoxins, including GIIIA; the class of calcium channel
antagonist ω-conotoxins, including GVIA, GVII, MVIIA, MVIIC,
and their analogs or synthetic derivatives; the class of NMDAantagonist conantokins, including con-G, con-R, con-T and their
analogs or synthetic derivatives; and the putative neurotensin
agonist, contulakin-G and its synthetic derivatives. Yersinia pestis
strains which are Pgm - due to a deletion of a 102-kb region of the
chromosome termed the pgm locus (i.e., ∆pgm). Examples are Y.
pestis strain E.V. or various substrains such as EV 76. Yersinia
pestis strains (e.g., Tjiwidej S and CDC A1122) devoid of the 75
kb low-calcium response (Lcr) virulence plasmid. Bacillus
anthracis strains devoid of both plasmids pX01 and pX02 &
Bacillus anthracis strains devoid of the plasmid pX02 (e.g.,
Bacillus anthracis Sterne, pX01+pX02-). Brucella abortus Strain
19 & Brucella abortus strain RB51 (vaccine strain). Coxiella
burnetii Phase II, Nine Mile Strain, plaque purified clone 4.
Francisella tularensis subspecies novicida (also referred to as
Francisella novicida) strain, Utah 112 (ATCC 15482) &
Francisella tularensis subspecies holartica LVS (live vaccine
strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684)
& Francisella tularensis ATCC 6223 (also known as strain B38).
Rift Valley fever virus, MP-12 vaccine strain. Venezuelan Equine
Encephalitis (VEE) virus vaccine candidate strain V3526 &
Venezuelan equine encephalitis virus, TC-83 strain. Highly
pathogenic avian influenza (HPAI) virus, recombinant vaccine
reference strains of the H5N1 and H5N3 subtypes. Japanese
encephalitis virus, SA14-14-2 strain..
The medical use of toxins for patient treatment is exempt.
USDA High Consequence Livestock Pathogen/Toxin
4
USDA-HHS Overlap Agent
9