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
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.
- 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.
- 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.
- 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 (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.
- 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.
- 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.
- 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.
- 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.
- 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 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.
- 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.
- Vodkin MH, Williams JC, Stephenson EH. Genetic heterogeneity among isolates of Coxiella burnetii. J. Gen. Microbiol. 1986 Feb; 132(2):455-63.
- 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.
- 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 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.
- 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.
- 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 subspecies novicida (also referred to as Francisella novicida) strain, Utah 112 (ATCC 15482) (effective 2-27-2003)
The exclusion is only for the type strain, Utah 112. This strain was originally isolated from a water sample taken from Ogden Bay, Utah in 1951. It is experimentally pathogenic for mice, guinea pigs and hamsters, producing lesions similar to those of tularemia; rabbits, white rats and pigeons are resistant. The Utah 112 strain is not known to infect man.
- Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol.Rev. 2002 Oct; 15(4): 631-46.
Francisella tularensis subspecies holarctica LVS (live vaccine strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684) (effective 2-27-2003)
This strain is used for studies on the genetics, biology, and pathogenesis of F. tularensis , studies on the host response to infection, development and use in diagnostic assays, and development of vaccines and therapeutics.
- Burke DS. Immunization against tularemia : analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis. 1977 Jan;135(1):55-60.
- Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol.Rev. 2002 Oct; 15(4): 631-46.
- Waag DM, Galloway A, Sandstrom G, Bolt CR, England MJ, Nelson GO, Williams JC. Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis. J Clin Microbiol. 1992 Sep;30(9):2256-64.
- Waag DM, McKee KT Jr, Sandstrom G, Pratt LL, Bolt CR, England MJ, Nelson GO, Williams JC. Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis. Clin Diagn Lab Immunol. 1995 Mar;2(2):143-8.
- Waag DM, Sandström G, England MJ, Williams JC. Immunogenicity of a new lot of Francisella tularensis live vaccine strain in human volunteers. FEMS Immunol Med Microbiol. 1996 Mar;13(3):205-9.
Francisella tularensis biovar tularensis strain B-38 (ATCC 6223) (effective 2-27-2003)
This strain has fastidious growth requirements and grows poorly in the laboratory. Mice are used as a model to study the pathogenesis of tularemia. The LD 50 of virulent strains of F. tularensis biovar tularensis for mice infected via the subcutaneous route is <10 CFU. However, mice infected intraperitoneally with 105 CFU or intradermally with 107 CFU of strain ATCC 6223 were not killed.
- Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol. Rev. 2002 Oct; 15(4):631-46.
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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.
- 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.
- 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.
- 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.
- 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.
- 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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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.
- Brubaker RR. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 1969 June; 98(3):1404-06.
- 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.
- 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.
- 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.
- 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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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).
- Hambleton P, Carman JA, Melling J. Anthrax: the disease in relation to vaccines. Vaccine. 1984 Jun; 2 (2):125-32.
- Shlyakhov EN , Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
- 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.
- Hambleton P, Camman JA, Melling J. Anthrax: The disease in relation to vaccines. Vaccine. 1984 Jun; 2(2):125-32.
- Shlyakhov EN, Rubinstein E. Human live anthrax vaccine in the former USSR. Vaccine. 1994 Jun; 12(8):727-30.
- Sterne M. Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind. 1946 Mar; 21:41-3.
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ΔnorDΔznuA Brucella abortus-lacZ (vaccine strain) (effective 06-02-2011)
The in-frame deletion of the genes znuA and norD that contribute to virulence of the wild-type pathogen prevents wild-type reversion. The znuA gene constitutes a high-affinity periplasmic binding protein-dependent and ATP-binding cassette (ABC) transport system for Zn2+(2-4). The norD gene is a member of the norEFCBQD operon encoding a nitric oxide reductase. This strain was shown to be attenuated in human and mouse macrophages.
- Beard SJ, Hashim R, Wu G, Binet MR, Hughes MN, Poole RK. Evidence for the transport of zinc (II) ions via the pit inorganic phosphate transport system in Escherichia coli. FEMS Microbiol Lett. 2000 Mar 15; 184(2):231-5.
- Kim S, Watanabe K, Shirahata T, Watarai M. Zinc uptake system ( znuA locus) of Brucella abortus is essential for intracellular survival and virulence in mice. J Vet Med Sci. 2004 Sep; 66(9):1059-63.
- Lewis DA, Klesney-Tait J, Lumbley SR, Ward CK, Latimer JL, Ison CA, Hansen EJ. Identification of the znuA -encoded periplasmic zinc transport protein of Haemophilus ducreyi. Infect Immun. 1999 Oct; 67(10):5060-8.
- Loisel-Meyer S, Jiménez de Bagüés MP, Bassères E , Dornand J, Köhler S, Liautard JP, Jubier-Maurin V. Requirement of norD for Brucella suis virulence in a murine model of in vitro and in vivo infection. Infect Immun. 2006 Mar; 74(3):1973-6.
- Yang X, Becker T, Walters N , Pascual DW. Deletion of znuA virulence factor attenuates Brucella abortus and confers protection against wild-type challenge. Infect Immun. 2006 Jul; 74(7):3874-9.
Brucella abortus S2308 phosphoglucomutase deletion mutant (Δpgm) (vaccine strain of Brucella abortus S2308) (effective 08/09/2006)
The vaccine strain known as B. abortus phosphoglucomutase deletion mutant (Δpgm) is a genetically modified strain with a 40% deletion of the pgm gene. The phosphoglucomutase mutant strain is less virulent than the parental strain 2308 with minimal colonization and persistence in the cranial lymph nodes and lack of abortion following conjunctival exposure in cattle. The vaccine strain does not synthesize the sugar-nucleotide UDP-sugars that proceed through a glucose-nucleotide intermediate. The strain is able to synthesize the O-polysaccharide, but is incapable of assembling complete lipopolysaccharides, due to the presence of an altered core structure.
- Elzer PH, Hagius SD, Davis DS, DelVecchio VG, Enright FM. Characterization of the caprine model for ruminant brucellosis. Vet Microbiol. 2002 Dec 20; 90(1-4):425-31.
- Ugalde JE, Comerci DJ, Leguizamón MS, Ugalde RA. Evaluation of Brucella abortus phosphoglucomutase ( pgm ) mutant as a new live rough-phenotype vaccine. Infect Immun. 2003 Nov; 71(11):6264-9.
- Ugalde JE, Czibener C , Feldman MF, Ugalde RA. Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication. Infect Immun. 2000 Oct; 68(10):5716-23.
Brucella abortus Strain 19 (effective 6-12-2003)
The Brucella abortus Strain 19 live vaccine, used in the U.S. Department of Agriculture Brucellosis Eradication Program from 1941 to 1996, is effective in the control of clinical brucellosis in cattle.
- Proceedings of the United States Animal Health Association 93:640-55.
- Joint FAO/WHO Expert Committee on Brucellosis. World Health Organization technical report series. 1986; 740:34-40.
- Young E. Human brucellosis. Review articles, Reviews of Infectious Diseases. 1983 Sept-Oct; 5(5):821-42
Brucella abortus strain RB51 (vaccine strain) (effective 5-7-2003)
Brucella abortus strain RB51 was conditionally licensed as a vaccine by USDA in 1996 and granted a full license in March 2003. It is used as part of the cooperative State-Federal Brucellosis Eradication Program. Brucella abortus strain RB51 is a genetically stable, rough morphology mutant of field strain Brucella. It lacks the polysaccharide O-side chains on the surface of the bacteria. Strain RB51 is less virulent than the Brucella abortus Strain 19 vaccine and field strain Brucella abortus.
- Brucellosis: http://www.aphis.usda.gov/animal_health/animal_diseases/brucellosis
- Schurig GG, Roop RM II, Bagchi T, Boyle S, Buhrman D, Sriranganathan N. Biological properties of RB51: a stable rough strain of Brucella abortus. Vet Microbiol 1991 Jul; 28(2):171-88.
- Stauffer B, Reppert J, Van Metre D, Fingland R, Kennedy G, Hansen G, Pezzino G, Olsen S, Ewalt D. Human exposure to Brucella abortus Strain RB51 – Kansas, 1997. MMWR 1998 Mar 13; 47(09):172-75.
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Burkholderia pseudomallei strain Bp82, a ΔpurM mutant of B. pseudomallei strain 1026b deficient in purine biosynthesis (effective 04-14-2010)
The B. pseudomallei ΔpurM mutant was shown to be fully attenuated in hyper susceptible animal models, including Syrian hamsters and 129/SvEv mice when infected via the inhalational challenge route. The mutant strain also failed to cause mortality in immune deficient mice. The mutant strain failed to replicate in vivo or disseminate following intranasal challenge. The attenuation of the strain was due to the ΔpurM defect since complementation of the Bp82 ΔpurM allele with wild-type sequence resulted in adenine prototrophy and restored virulence.
- Propst KL, Mima T, Choi KH , Dow SW, Schweizer HP. A Burkholderia pseudomallei ΔpurM mutant is avirulent in immunocompetent and immunodeficient animals: candidate strain for exclusion from select-agent lists. Infect Immun. 2010 Jul; 78(7):3136-43.
Burkholderia pseudomalleis strain B0011, a Δasd mutant of B. pseudomallei strain 1026b (effective 12-07-2011)
This strain contains a deletion in the aspartate-B-semialdehyde dehydrogenase ( asd ) gene, which is auxotrophic for diaminopimelate (DAP). The Δ asd mutant was found to be avirulent in mice and unable to replicate in HeLa or RAW 264.7 cells.
- Norris MH, Propst KL, Kang Y, Dow SW, Schweizer HP, Hoang TT. The Burkholderia pseudomallei Δ asd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice. Infect Immun. 2011 Oct; 79(10):4010-8.
- Norris MH, Kang Y, Lu D, Wilcox BA, Hoang TT. Glyphosate resistance as a novel select-agent-compliant, non-antibiotic-selectable marker in chromosomal mutagenesis of the essential genes asd and dapB of Burkholderia pseudomallei. Appl Environ Microbiol. 2009 Oct; 75(19):6062-75.
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RIFT VALLEY FEVER VIRUS
Rift Valley fever (RVF) virus vaccine strain MP-12 (effective 2-7-2003)
The MP-12 attenuated strain of RVF virus was obtained by 12 serial passages of a virulent isolate ZH548 in the presence of 5-fluorouracil. Hamsters infected with the ZH548-M12 vaccine candidate strains survived and were immune to challenge with 105 of the wild-type ZH501 strain of RVF virus. The attenuated strain also failed to replicate in Vero cells at 41°C. Studies have also shown that MP-12 protects the bovine and ovine dam and fetus against virulent viral challenge and is safe and efficacious for use in neonatal calves and lambs.
- Caplen H, Peters CJ, Bishop DH. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J Gen Virol. 1985 Oct; 66 (10):2271-7.
- Hubbard KA, Baskerville A, Stephenson JR. Ability of a mutagenized virus variant to protect young lambs from Rift Valley fever. Am J Vet Res. 1991 Jan; 52(1):50-5.
- Morrill JC, Mebus CA, Peters CJ. Safety and efficacy of a mutagen-attenuated Rift Valley fever virus vaccine in cattle. Am J Vet Res. 1997 Oct; 58(10):1104-9.
- Morrill JC, Peters CJ. Pathogenicity and neurovirulence of a mutagen-attenuated Rift Valley fever vaccine in rhesus monkeys. Vaccine. 2003 Jun 20; 21(21-22):2994-02.
- Morrill JC, Jennings GB, Caplen H, Turell MJ, Johnson AJ, Peters CJ. Pathogenicity and immunogenicity of a mutagen-attenuated Rift Valley fever virus immunogen in pregnant ewes. Am J Vet Res. 1987 Jul; 48(7):1042-7.
- Morrill JC, Carpenter L, Taylor D, Ramsburg HH, Quance J, Peters CJ. Further evaluation of a mutagen-attenuated Rift Valley fever vaccine in sheep. Vaccine. 1991 Jan; 9(1):35-41.
- Morrill JC, Mebus CA, Peters CJ. Safety of a mutagen-attenuated Rift Valley fever virus vaccine in fetal and neonatal bovids. Am J Vet Res. 1997 Oct; 58(10):1110-4.
- Rossi CA, Turell MJ. Characterization of attenuated strains of Rift Valley fever virus. J Gen Virol. 1988 Apr; 69(4):817-23.
- Saluzzo JF, Smith JF. Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine. Vaccine. 1990 Aug; 8(4):369-75.
- Turell MJ, Rossi CA. Potential for mosquito transmission of attenuated strains of Rift Valley fever virus. Am J Trop Med Hyg. 1991 Mar; 44(3):278-82.
- Vialat P, Muller R, Vu TH , Prehaud C , Bouloy M. Mapping of the mutations present in the genome of the Rift Valley fever virus attenuated MP12 strain and their putative role in attenuation. Virus Res. 1997 Nov; 52(1):43-50.
Live-attenuated Rift Valley fever virus vaccine candidate strain ΔNSs-ΔNSm-ZH501 (effective 03-12-2012)
The strain is attenuated and cannot be transmitted by mosquito. There is no evidence that it causes viremia in animal models (rats, mice, non-human primates, sheep, or pregnant sheep). Further, whole gene deletions make it difficult to regenerate two virulence components.
- Bird BH, Albariño CG, Hartman AL, Erickson BR, Ksiazek TG, Nichol ST. Rift valley fever virus lacking the NSs and NSm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals. J Virol. 2008 Mar; 82(6):2681-91.
- Bird BH, Maartens LH, Campbell S, Erasmus BJ, Erickson BR, Dodd KA, Spiropoulou CF, Cannon D, Drew CP, Knust B, McElroy AK, Khristova ML, AlbariñoCG, Nichol ST. Rift Valley fever virus vaccine lacking the NSs and NSm genes issafe, nonteratogenic, and confers protection from viremia, pyrexia, and abortion following challenge in adult and pregnant sheep. J Virol. 2011Dec; 85(24):12901-9.
- Crabtree MB, Kent Crockett RJ, Bird BH, Nichol ST, Erickson BR, Biggerstaff BJ, Horiuchi K, Miller BR. Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection. PLoS Negl Trop Dis. 2012 May; 6(5):e1639.
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VENEZUELAN EQUINE ENCEPHALITIS
Venezuelan equine encephalitis (VEE) subtypes ID and IE (effective 12-4-2012)
The VEEV virus strains designated enzootic are those belonging the ID and IE varieties. The reasons for excluding ID and IE VEEVs from the select agent list are: (1) No subtype ID and IE VEEV have ever been documented to cause large equine epizootics; (2) While ID strains are the ancestral forms of the IC variety, inclusion of ID viruses because they might be precursors to IC viruses is not sufficient justification for making ID viruses select agents. The possibility of a ID virus mutating to a IC virus following a bioterrorism event is unlikely because ID viruses are unlikely to establish epidemic or epizootic transmission cycles in the U.S. Natural transmission cycles requiring specific mosquito vectors would likely be needed for any evolution from ID to IC to occur in nature in the US; and (3) The currently available humanized or human anti-VEEV monoclonal antibodies that could be produced for emergency use could also have prophylactic, and possibly therapeutic efficacy for all VEEV subtype 1 infections with which they cross react (includes ID and IE viruses). Straightforward diagnostic molecular techniques [such as sequencing or RT-PCR (reverse transcription - polymerase chain reaction) with subtype/variety specific primer sets] or serological testing with specific monoclonal antibodies, can distinguish between enzootic (ID and IE) and epizootic (IAB and IC) VEE strains.
- Hart MK, Lind C, Bakken R, Robertson M, Tammariello R, Ludwig GV. Onset and duration of protective immunity to IA/IB and IE strains of Venezuelan equine encephalitis virus in vaccinated mice. Vaccine. 2001 Nov 12;20(3-4):616-22.
- Phillpotts RJ , Jones LD , Howard SC. Monoclonal antibody protects mice against infection and disease when given either before or up to 24 h after airborne challenge with virulent Venezuelan equine encephalitis virus. Vaccine. 2002 Feb 22;20(11-12):1497-504.
- Schmaljohn AL, Johnson ED, Dalrymple JM, Cole GA. Non-neutralizing monoclonal antibodies can prevent lethal alphavirus encephalitis. Nature. 1982 May 6;297(5861):70-2.
- Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC. Venezuelan equine encephalitis. Annu Rev Entomol. 2004; 49:141-74.
- Meissner JD, Huang CY, Pfeffer M, Kinney RM. Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex. Virus Res. 1999 Oct;64(1):43-59.
- Oberste MS, Weaver SC, Watts DM, Smith JF. Identification and genetic analysis of Panama-genotype Venezuelan equine encephalitis virus subtype ID in Peru. Am J Trop Med Hyg. 1998 Jan;58(1):41-6.
- Oberste MS, Schmura SM, Weaver SC, Smith JF. Geographic distribution of Venezuelan equine encephalitis virus subtype IE genotypes in Central America and Mexico. Am J Trop Med Hyg. 1999 Apr;60(4):630-4.
- O'Brien LM, Goodchild SA, Phillpotts RJ, Perkins SD. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge. Virology. 2012 May 10;426(2):100-5. doi: 10.1016/j.virol.2012.01.038. Epub 2012 Feb 15.
Venezuelan Equine Encephalitis (VEE) virus vaccine candidate strain V3526 (effective 5-5-2003)
VEE strain V3526 is an attenuated strain of VEE, which was constructed by site-directed mutagenesis. V3526 contains two mutations relative to the virulent parental clone. One of these mutations is a deletion, which renders the virus non-viable; the other mutation restores viability without restoring the pathogenic properties of the parental virus. The stability of the deletion mutation in V3526 fundamentally and significantly decreases the hazard associated with this strain, and makes it unlikely that it can revert to wild type. This strain is considerably less virulent than the excluded vaccine strain TC83.
- Davis NL, Brown KW, Greenwald GF, Zajac AJ, Zacny VL, Smith JF. Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second site suppressor mutation in E1. Virology. 1995 Sep; 212(1):102-10.
Venezuelan Equine Encephalitis (VEE) virus vaccine strain TC-83 (effective 2-7-2003)
Mice vaccinated subcutaneously with the attenuated vaccine strain of VEE virus rapidly developed immunity to subcutaneous or airborne challenge with virulent VEE virus.
- Razumov IA, Agapov EV, Pereboev AV, Protopopova EV, Lebedeva SD, Loktev VB. Investigation of antigenic structure of attenuated and virulent Venezuelan equine encephalomyelitis virus by means of monoclonal antibodies. Biomed Sci. 1991; 2(6):615-22
- Phillpotts RJ, Wright AJ. TC-83 vaccine protects against airborne or subcutaneous challenge with heterologous mouse-virulent strains of Venezuelan equine encephalitis virus. Vaccine. 1999 Feb 26; 17(7-8):982-8.
- Phillpotts RJ. Immunity to airborne challenge with Venezuelan equine encephalitis virus develops rapidly after immunization with the attenuated vaccine strain TC-83. Vaccine. 1999 May 14; 17(19):2429-35.
- Bennett AM, Elvin SJ, Wright AJ, Jones SM, Phillpotts RJ. An immunological profile of Balb/c mice protected from airborne challenge following vaccination with a live attenuated Venezuelan equine encephalitis virus vaccine. Vaccine. 2000 Sep 15; 19(2-3):337-47.
- Elvin SJ, Bennett AM, Phillpotts RJ. Role for mucosal immune responses and cell-mediated immune functions in protection from airborne challenge with Venezuelan equine encephalitis virus. J Med Virol. 2002 Jul; 67(3):384-93.
VRPs constructed using the V3014 derived helper of Venezuelan Equine Encephalitis (VEE) virus (effective 12-29-2004)
Batch tests negative using the FDA approved test for detecting Replication Competent Virus (RCV).
- Pushko P, Parker M, Ludwig GV, Davis NL, Johnston RE, Smith JF. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology. 1997 Dec 22; 239(2):389-01.
Sindbis/VEE virus and Sindbis/Eastern Equine Encephalitis (EEE) virus chimeric constructions that include the structural genes (only) of VEE virus or EEE virus (effective 5-29-2007)
Chimeras derived from EEE virus strains FL93-939 (Sin/Pl93-939) and BeAR436087 (Sin/BeAr436087), and VEE virus strains Trinidad donkey (Sin/TRD), TC83 (Sin83), and ZPC738 (Sin/ZPC) are excluded. These constructs have been tested in animal models and have shown to be attenuated. The exclusion does not include chimeras derived from other VEE virus or EEE virus strains or chimeras including additional VEE virus or EEE virus components.
- Aguilar PV, Paessler S, Carrara AS, Baron S, Poast J, Wang E, Moncayo AC, Anishchenko M, Watts D, Tesh RB, Weaver SC. Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus. J Virol. 2005 Sep; 79(17):11300-10.
- McKnight KL, Simpson DA, Lin SC, Knott TA, Polo JM, Pence DF, Johannsen DB, Heidner HW, Davis NL, Johnston RE. Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes. J Virol. 1996 Mar; 70(3):1981-9.
- Lustig S, Jackson AC, Hahn CS, Griffin DE, Strauss EG, Strauss JH. Molecular basis of Sindbis virus neurovirulence in mice. J Virol. 1988 Jul; 62(7):2329-36.
- Paessler S, Fayzulin RZ, Anishchenko M, Greene IP, Weaver SC, Frolov I. Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic. J Virol. 2003 Sep; 77(17):9278-86.
- Paessler S, Ni H, Petrakova O, Fayzulin RZ, Yun N, Anishchenko M, Weaver SC, Frolov I. Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses. J Virol. 2006 Mar; 80(6):2784-96.
- Rice CM, Levis R, Strauss JH, Huang HV. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec; 61(12):3809-19.
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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) 103KB. Specific reference vaccine strains have not been listed here for proprietary reasons.
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