National Select Agent Registry phone numbers for APHIS (301-851-3300) and CDC (404-718-2000).
Last Updated: Thursday, January 16, 2014

Select Agent and Toxin Exclusions

The select agent regulations (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73) established a procedure by which an attenuated strain of a select biological agent or toxin that does not pose a severe threat to public health and safety, animal health, or animal products may be excluded from the requirements of the select agent regulations.

Final Rule (October 5, 2012)
On October 5, 2012, the final rule excluded any low pathogenic strains of avian influenza virus, any strain of Newcastle disease virus which does not meet the criteria for virulent Newcastle disease virus, all subspecies Mycoplasma capricolum except subspecies capripneumoniae (contagious caprine pleuropneumonia), and all subspecies Mycoplasma mycoides except subspecies mycoides small colony (Mmm SC) (contagious bovine pleuropneumonia), provided that the individual or entity can verify that the agent is within the exclusion category.

In addition, the final rule included the removal of the South American genotypes of Eastern Equine Encephalitis virus (EEE), all Venezuelan Equine Encephalitis virus (VEE) subtypes except IAB and IC, the West African clade of Monkeypox virus and all conotoxins except the sub-class of conotoxins generally called "short, paralytic alpha conotoxins," exemplified by α-conotoxin GI and α-conotoxin MI and containing the following amino acid sequence X1CCX2 PACGX3X4X5X6CX7.

To prevent confusion on how an entity should handle samples that have been determined to be within a general taxonomic classification ( e.g ., EEE) but not within a particular genotype or subtype ( e.g., North American EEE virus ), the current general taxonomic listing of HHS and overlap select agents was maintained as opposed to listing a specific strain and adding an exclusion for the strains, subtypes, or pathogenicity levels which are not considered to have the potential to pose a severe threat to public health and safety. When an agent is initially identified by taxonomic classification, it is subject to the select agent regulations until further testing is accomplished to exclude the particular agent by strain, subtype, or pathogenicity level.

North American EEE virus (NA-EEE) genotype strains, which are the strains responsible for human and equine disease, are all genetically very similar to each other (less than 3 percent divergence at the nucleotide level) and can be easily distinguished from South American EEE virus (SA-EEE) genotype strains using diagnostic molecular techniques.

We also note that there are published diagnostic tests that differentiate the Congo Basin clade of Monkeypox virus from the West African clade.

Attenuated Strains of HHS and USDA Select Agents and Toxins

Based upon consultations with subject matter experts and a review of relevant published studies and information provided by the entities requesting the exclusions, the Federal Select Agent Program has determined that the following attenuated strains or toxin proteins are not subject to the requirements of the select agent regulations if used in basic or applied research, as positive controls, for diagnostic assay development, or for the development of vaccines and therapeutics.

However, an individual or entity that possesses, uses, or transfers an excluded attenuated strain will be subject to the regulations if there is any reintroduction of factor(s) associated with virulence or other manipulations that modify the attenuation such that virulence is restored or enhanced. In addition, attenuated strains that are excluded from the requirements of the select agent regulations are not exempt from the requirements of other applicable regulations or guidelines (e.g., NIH guidelines, USDA/APHIS permits, etc.).

Attenuated strains of HHS Select Agents and Toxins excluded Attenuated strains of Overlap Select Agents excluded Attenuated strains of USDA-only select agents excluded

Attenuated strains of HHS Select Agents and Toxins excluded from the requirements of 42 CFR part 73:

BOTULINUM NEUROTOXIN

Botulinum neurotoxin proteins

  • Fusion proteins of the heavy-chain domain of BoNT/translocation domain of diphtheria toxin (effective 07-28-2011)
    Fusion proteins consisting of the heavy-chain domain of BoNT and the translocation domain of diphtheria toxin (no catalytic domain); however, the reconstitution of the BoNT holotoxin would be considered a select toxin.

    Reference(s):
    1. Ho M, Chang LH, Pires-Alves M, Thyagarajan B, Bloom JE, Gu Z, Aberle KK, Teymorian SA, Bannai Y, Johnson SC, McArdle JJ,Wilson BA. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting. Protein Engineering, Design and Selection. 2011 Mar; 24(3):247-53.

  • BoNT purified protein (BoNT/A1 atoxic derivative, ad, E224A/Y366A) (effective 07-22-2009)
    BoNT purified protein (BoNT/A1 atoxic derivative, ad, E224A/Y366A) that has been expressed from recombinant DNA constructs is non-catalytic and non-toxic as a result of a double mutation introduced into the region of the gene encoding the light chain. This exclusion is for the protein only and not for the recombinant DNA construct of BoNT/A1 encoding the ad protein.

  • Recombinant Botulinum neurotoxin serotype A (R362A, Y365F) (effective 03-28-2006)
    Recombinant Botulinum neurotoxin serotype A (R362A, Y365F), termed BoNT/A(RY), is a product of two amino acid substitutions engineered into the light chain of Botulinum neurotoxin serotype A (BoNT/A). Arg362Ala (three base substitution) and Tyr365Phe (two base substitution) yields a protein that does not cleave SNAP25 (the mutated toxin is >100-fold less catalytic than native protein). One microgram of recombinant Botulinum neurotoxin serotype A BoNT/A (RY) is not toxic in the mouse model of BoNT intoxication.

    Reference(s):
    1. Rossetto O, Caccin P, Rigoni M, Tonello F, Bortoletto N, Stevens RC, Montecucco C. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity. Toxicon. 2001 Aug; 39(8):1151-9.
    2. Barbieri JT, Collier RJ. Expression of a mutant, full-length form of diphtheria toxin in Escherichia coli. Infect Immun. 1987 Jul; 55(7):1647-51.

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CONOTOXINS

  • Conotoxins (non-short, paralytic alpha conotoxins; effective 12-4-2012)
    Based upon available experimental evidence, most known conotoxins (i.e.,"conopeptides") do not possess sufficient acute toxicity to pose a significant public health threat, and many are employed as useful research tools or potential human therapeutics. However, currently available data demonstrate that the sub-class of conotoxins generally called "short, paralytic alpha conotoxins," exemplified by α-conotoxin GI and α-conotoxin MI, do possess sufficient acute toxicity by multiple routes of exposure, biophysical stability, ease of synthesis, and availability. The conotoxins that remain on the HHS list will be limited to the short, paralytic alpha conotoxins containing the following amino acid sequence X1 CCX2 PACGX3 X4 X5 X6 CX7, whereas:
    • The consensus sequence includes known toxins α-MI and α-GI (shown above) as well as α-GIA, Ac1.1a, α-CnIA, α-CnIB;
    • C = Cysteine residues are all present as disulfides, with the 1st and 3rd Cysteine, and the 2nd and 4th Cysteine forming specific disulfide bridges;
    • X1 = any amino acid(s) or Des-X;
    • X2 = Asparagine or Histidine;
    • P = Proline;
    • A = Alanine;
    • G = Glycine;
    • X3 = Arginine or Lysine;
    • X4 = Asparagine, Histidine, Lysine, Arginine, Tyrosine, Phenylalanine or Tryptophan;
    • X5 = Tyrosine, Phenylalanine, or Tryptophan;
    • X6 = Serine, Threonine, Glutamate, Aspartate, Glutamine, or Asparagine;
    • X7 = Any amino acid(s) or Des X;
    • "Des X" = "an amino acid does not have to be present at this position." For example if a peptide sequence were XCCHPA then the related peptide CCHPA would be designated as Des-X.

    The short, paralytic alpha conotoxins containing the following amino acid sequence X1 CCX2 PACGX3 X4 X5 X 6 CX 7 will be considered a select toxin if the total amount (all forms) under the control of a principal investigator, treating physician or veterinarian, or commercial manufacturer or distributor exceeds 100 mg at any time.

    Reference(s):
    1. Favreau P, Krimm I, Le Gall F, Bobenrieth MJ, Lamthanh H, Bouet F, Servent D, Molgo J, Ménez A, Letourneux Y, Lancelin JM. Biochemical characterization and nuclear magnetic resonance structure of novel α -conotoxins isolated from the venom of Conus consors. Biochemistry. 1999 May 11; 38(19):6317-26.
    2. Groebe DR, Dumm JM, Levitan ES, Abramson SN. alpha-Conotoxins selectively inhibit one of the two acetylcholine binding sites of nicotinic receptors. Mol Pharmacol. 1995 Jul; 48(1):105-11.
    3. Groebe DR, Gray WR, Abramson SN. Determinants involved in the affinity of α -conotoxins GI and SI for the muscle subtype of nicotinic acetylcholine receptors. Biochemistry. 1997 May 27; 36(21):6469-74.
    4. Liu L, Chew G, Hawrot E, Chi C, Wang C. Two potent alpha3/5 conotoxins from piscivorous Conus achatinus. Acta Biochim Biophys Sin (Shanghai). 2007 Jun; 39(6):438-44.
    5. Stiles BG. Acetylcholine receptor binding-characteristics of snake and cone snail venom postsynaptic neurotoxins: further studies with a non-radiological assay. Toxicon. 1993 Jul; 31(7):825-34.

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COXIELLA BURNETTI

  • Coxiella burnetii Phase II, Nine Mile Strain, plaque purified clone 4 (effective 10-15-2003)
    Lipopolysaccharide (LPS) phase variation is the only confirmed virulence factor of C. burnetii. Organisms isolated from natural infections or in the laboratory are in phase I and have a smooth-type LPS. Repeated passage of phase I organisms through embryonated eggs or cultured cells resulted in the conversion to phase II and a change in the LPS to a rough-type. Injection of such laboratory-derived phase II variants into guinea pigs resulted in infection and reversion to phase I. However, plaque-purified (cloned) isolates of the Nine Mile Strain phase II organisms do not undergo phase reversion and are avirulent since inoculation of susceptible animals with phase II cells does not result in infection nor can viable phase II or phase I organisms be recovered from the spleens of these animals. The Nine Mile Strain plaque purified phase II is stable and does not revert to phase I; restriction fragment-length polymorphisms detected after Hae -III digestion of chromosomal DNA and DNA-DNA hybridization, suggests that the Nine Mile Strain plaque purified phase II variant has undergone a deletion. Based upon consultations with subject matter experts and a review of relevant published studies, HHS and USDA have determined that Coxiella burnetii, Phase II, Nine Mile Strain, plaque purified clone 4, does not pose a significant threat to human or animal health.

    Reference(s):
    1. O'Rourke AT, Peacock M, Samuel JE, Frazier ME, Natvig DO, Mallavia LP, Baca O. Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases. J. Gen. Microbiol. 1985 June; 131(6):1543-46.
    2. Vodkin MH, Williams JC, Stephenson EH. Genetic heterogeneity among isolates of Coxiella burnetii. J. Gen. Microbiol. 1986 Feb; 132(2):455-63.
    3. Moos A, Hackstadt T. Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model. Infect. Immun. 1987 May; 55(5):1144-50.

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EASTERN EQUINE ENCEPHALITIS VIRUS (EEEV)

  • South American genotypes of Eastern Equine Encephalitis virus (effective 12-4-2012)
    The final rule excluded the South American genotypes of Eastern Equine Encephalitis virus (EEE). North American EEE virus (NA-EEE) genotype strains, which are the strains responsible for human and equine disease. T he factors that we considered in retaining the NA EEEV genotype were that this genotype exhibits high morbidity, high mortality, and has the potential to be weaponized. In contrast, South American strains are typically avirulent for humans and are not clearly linked to human disease. Further distinctions between these strains and those from North America have led to the suggestion that these are actually 2 distinct viruses and that the South American EEEV strains should not be considered select agents.

    Reference(s)
    1. Arrigo NC, Adams AP, Weaver SC. Evolutionary patterns of eastern equine encephalitis virus in North versus South America suggest ecological differences and taxonomic revision. J Virol. 2010 Jan; 84(2):1014-25.

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EBOLA VIRUS

  • Ebola ΔVP30 replication incompetent virus (effective 01-02-2013)
    This virus lacks the gene encoding for the VP30 protein; therefore, biologically contained Ebola virus (Ebola ΔVP30 viruses) are replication incompetent and do not form infectious progeny in wild-type cells due to the lack of the VP30 gene. The genome of Ebola ΔVP30 virus is stable, infection of Vero cells and various animals with Ebola ΔVP30 virus particles did not indicate a single event of virus replication, infection of animals with Ebola ΔVP30 virus particles did not cause disease in infected animals.

    Reference(s)
    1. Halfmann P, Kim JH , Ebihara H , Noda T, Neumann G , Feldmann H , Kawaoka Y. Generation of biologically contained Ebola viruses. Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1129-33.
    2. Halfmann P, Ebihara H, Marzi A, Hatta Y, Watanabe S, Suresh M, Neumann G, Feldmann H, Kawaoka Y. Replication-deficient ebolavirus as a vaccine candidate. J Virol. 2009 Apr;83(8):3810-5.

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FRANCISELLA TULARENSIS

JUNIN VIRUS

  • Junin virus vaccine strain Candid No. 1 (effective 02-07-2003)
    The protective efficacy of Candid No. 1, a live-attenuated vaccine against Argentine hemorrhagic fever (AHF), was evaluated in non-human primates. Twenty rhesus macaques immunized 3 months previously with graded doses of Candid No. 1 (16-127,000 PFU), as well as 4 placebo-inoculated controls, were challenged with 4.41 log 10 PFU of virulent P3790 strain Junin virus. All controls developed severe clinical disease; 3 of 4 died. In contrast, all vaccinated animals were fully protected; none developed any signs of AHF during a 105 day follow-up period. Candid No. 1 was highly immunogenic and fully protective against lethal virus challenge in rhesus macaques, even at extremely low (16 PFU) vaccine doses.

    Reference(s):
    1. McKee KT Jr, Oro JG, Kuehne AI, Spisso JA, Mahlandt BG. Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junin virus challenge in rhesus macaques. Intervirology. 1992: 34(3):154-63.


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LASSA FEVER VIRUS

  • Mopeia/Lassa (MOP/LAS) arenavirus construct ML-29 (effective 03-02-2005)
    The construct is not capable of encoding infectious and/or replication competent forms of any select agent viruses. Clone ML29, selected from a library of MOPV/LASV (MOP/LAS) reassortants, encodes for the major antigens (nucleocapsid and glycoprotein) of LASV and the RNA polymerase and zinc-binding protein of MOPV. Replication of clone ML29 was attenuated in guinea pigs and nonhuman primates. In murine adoptive-transfer experiments, as little as 150 PFU of clone ML29 induced protective cell-mediated immunity. All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions. Rhesus macaques inoculated with clone ML29 developed primary virus-specific T cells capable of secreting gamma interferon in response to homologous MOP/LAS and heterologous MOPV and lymphocytic choriomeningitis virus. Detailed examination of two rhesus macaques infected with this MOPV/LAS reassortant revealed no histological lesions or disease signs.

    Reference(s):
    1. Kiley MP, Lange JV, Johnson KM. Protection of rhesus monkeys from Lassa virus by immunization with closely related Arenavirus. Lancet. 1979 Oct 6; 314(8145):738.
    2. Lange JV, Mitchell SW, McCormick JB, Walker DH, Evatt BL, Ramsey RR. Kinetic study of platelets and fibrinogen in Lassa virus -infected monkeys and early pathologic events in Mopeia virus -infected monkeys. Am J Trop Med Hyg. 1985 Sep; 34(5):999-1007.
    3. Lukashevich IS, Patterson J, Carrion R, Moshkoff D, Ticer A, Zapata J, Brasky K, Geiger R, Hubbard GB, Bryant J, Salvato MS. A live attenuated vaccine for Lassa fever made by reassortment of Lassa and Mopeia viruses. J Virol. 2005 Nov; 79(22):13934-42.
    4. Peters CJ , Jahrling PB , Liu CT, Kenyon RH, McKee KT Jr, Barrera Oro JG. Experimental studies of arenaviral hemorrhagic fevers. Curr Top Microbiol Immunol. 1987; 134:5-68.

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MONKEYPOX VIRUS

YERSINIA PESTIS

  • Yersinia pestis strains which are Pgm- due to a deletion of a 102-kb region of the chromosome termed the pgm locus (i.e., Δpgm). Examples are Y. pestis strain EV or various substrains such as EV 76 (effective 3-14-2003)
    Pgm- mutants of Yersinia pestis occur at a high frequency (ca 10-5) and result in avirulence and Pgm- strains such as the EV 76 strain. These strains have been used for years as live human vaccines with no significant plague-associated problems. The mutation in question is due to the excision of about 102-kb of chromosomal DNA via reciprocal recombination between adjacent IS 100 elements. The lost DNA sequence encodes the ability to synthesize and utilize the siderophore yersiniabactin, which is necessary for growth in mammalian peripheral tissue, as well as the Hms+ locus, which is necessary for biofilm production in the flea vector. However, PCR and/or Southern blot analysis will be required to ensure that "Pgm- " derivatives have undergone this deletion rather than a mutation in the hemin storage genes (hms), which also causes loss of Congo red (CR) binding, which is the most common characteristic used to evaluate the pigmentation phenotype.

    Reference(s):
    1. Brubaker RR. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 1969 June; 98(3):1404-06.
    2. Fetherston JD, Scheutze P, Perry RD. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol. Microbiol. 1992 Sept; 6(18):2693-04.
    3. Bearden SW, Perry RD. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol. Microbiol. 1999 Apr; 32(2):403-14.
    4. Une, T, Brubaker BB. In vivo comparison of avirulent Vwa - and Pgm - or Pstr phenotypes of Yersiniae. Infect. Immun.1984 Mar; 43(3):895-00.

  • Yersinia pestis strains (e.g., Tjiwidej S and CDC A1122) devoid of the 75 kb low-calcium response (Lcr) virulence plasmid (effective 2-27-2003)
    Strains of Yersinia pestis that lack the 75 kb low-calcium response (Lcr) virulence plasmid are excluded. Strains lacking the Lcr plasmid (Lcr-) are irreversibly attenuated due to the loss of a virulence plasmid. An Lcr- strain of Yersinia pestis (Tjiwidej S) has been extensively used as a live vaccine in humans in Java.

    Reference(s):
    1. Meyer KF, Cavanaugh DC, Bartelloni PJ, Marshal JD Jr. Plague immunization. I. Past and present trends. J. Infect. Dis. 1974 May; 129 (suppl1): S13-S18.


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Attenuated strains of Overlap Select Agents excluded from the requirements of 9 CFR Part 121 and 42 CFR part 73:

BACILLUS ANTHRACIS

  • Bacillus anthracis strains devoid of both plasmids pX01 and pX02. (effective 2-27-2003)
    Bacillus anthracis strains devoid of both virulence plasmids pX01 and pX02 are excluded based on published studies evaluating the attenuation of strains containing different combinations of the two plasmids).

    Reference(s):
    1. Hambleton P, Carman JA, Melling J. Anthrax: the disease in relation to vaccines. Vaccine. 1984 Jun; 2 (2):125-32.
    2. Shlyakhov EN , Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
    3. Sterne M. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind. 1946 Mar; 21:41-3.

  • Bacillus anthracis strains devoid of the plasmid pX02 (e.g., Bacillus anthracis Sterne, pX01+ pX02 -). (effective 2-27-2003)
    Bacillus anthracis strains lacking the virulence plasmid pX02 (e.g., Sterne, pX01+ and pX02 -) indicate that these strains were 105 to 107 fold less virulent than isogenic strains with both plasmids. These strains have been used to vaccinate both humans and animals.

    Reference(s):
    1. Hambleton P, Camman JA, Melling J. Anthrax: The disease in relation to vaccines. Vaccine. 1984 Jun; 2(2):125-32.
    2. Shlyakhov EN, Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
    3. Sterne M. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind. 1946 Mar; 21:41-3.

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BRUCELLA ABORTUS

BURKHOLDERIA PSEUDOMALLEI

RIFT VALLEY FEVER VIRUS

VENEZUELAN EQUINE ENCEPHALITIS

Attenuated strains of USDA-only select agents excluded from the requirements of 9 CFR Part 121

AVIAN INFLUENZA VIRUS (Highly Pathogenic)

  • Avian influenza virus (highly pathogenic), recombinant vaccine reference strains of the H5N1 and H5N3 subtypes (effective 5-7-2004)
    Several recombinant reference vaccine strains of highly pathogenic subtypes have been excluded based on results from in-vitro and in-vivo studies indicating that these strains were not pathogenic in avian species. The data requirements necessary for exclusion consideration under 9 CFR 121.3(g)PDF version 103KB. Specific reference vaccine strains have not been listed here for proprietary reasons.


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