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

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.

Burkholderia mallei Δasd strains (effective 12-13-2017)

The asd gene, encoding the aspartate semi-aldehyde dehydrogenase gene, is deleted in all B. mallei strains listed in the table above. The asd mutants lack the ability to cross-link their peptidoglycan cell-wall and, in the absence of diaminopimelic acid (DAP), will die due to osmotic stress. DAP is a bacterial specific metabolite that is not made in hosts including mammals; therefore, these mutants are completely attenuated in vivo and are unable to replicate within the host.

Reference(s):

1. 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.
2. 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.

Burkholderia mallei strain CLH001, a ∆tonB ∆hcp1 mutant of B. mallei strain ATCC 23344 (potential vaccine candidate strain) (effective August 20, 2015)
B. mallei strain CLH001 was derived from the B. mallei tonB mutant (TMM001) deficient in iron acquisition.  The tonB gene encodes a periplasmic protein required for siderophore-mediated iron uptake. In addition, the strain contains a 162 base pair intragenic in-frame deletion of the hemolysin co-regulated protein-1 (hcp1) gene, which encodes for a protein required for the assembly and functionality of the type six secretion system cluster 1 (T6SS-1). The construction of a double deletion mutant eliminates the possibility of reversion to wild-type virulence. The CLH001 strain was administered to BALB/c mice via a single intranasal dose of 1.5×104 CFU or via three intranasal doses (administered bi-weekly) of either 1.5×104 or 1.5×105 CFU. The mice exhibited 100% survival (unpublished data). In addition, immunocompromised mice (NSG) were challenged with 1.5×104 CFU of CLH001. After 21 days, the immunocompromised mice did not display any overt signs of infection and exhibited 100% survival. The strain could not be recovered from the lungs, liver, or spleen.

Reference(s):

1. Mott TM, et al., Characterization of the Burkho/deria ma//el tonB Mutant and its potential as a backbone strain for vaccine development. PLoS Neg/ Trop Dis 2015. [In press].
2. Burtnick MN, DeShazer D, Nair V, Gherardini FC, Brett PJ. Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun. 2010 Jan;78(1):88-99.
3. Schell MA, Ulrich RL, Ribot WJ, Brueggemann EE, Hines HB, Chen D, Lipscomb L, Kim HS, Mrázek J, Nierman WC, Deshazer D. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol. 2007 Jun;64(6):1466-85=
4. Lever MS, Nelson M, Ireland PI, Stagg AJ, Beedham RJ, Hall GA, Knight G, Titball RW. Experimental aerogenic Burkholderia mallei (glanders) infection in the BALB/c mouse. J Med Microbiol. 2003 Dec;52(Pt 12):1109-15.
5. Massey S, Johnston K, Mott TM, Judy BM, Kvitko BH, Schweizer HP, Estes DM, Torres AG. In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model.Front Microbiol. 2011 Aug 26;2:174.

Burkholderia pseudomallei strain PBK001 (ΔtonB Δhcp1) (effective 01-14-2020)
Burkholderia pseudomallei (Bp)PBK001 strain contains a deletion of the tonBgene, which encodes a periplasmic protein required for siderophore-mediated iron uptake. Iron uptake is important for virulence and establishing an infection in the host. The strain also contains a deletion in the hcp1 gene, which is a component of the type six secretion system utilized by the bacteria for cell-to-cell spread and dissemination. Mice (n=15) were administered a single intranasal dose of 1.5×10⁵ CFU of the Bp PBK001 strain (equivalent to 100 LD₅₀ of wild-type Bp K96243) and the survival was monitored for 28 days. The mice exhibited 100% survival and the bacteria disappeared from the lungs within 2 days of the infection. No bacteria were detected in any of the other target organs (liver and spleen). No symptoms of overt infection (ruffled fur, hunched posture, weight loss, etc.) were observed in these animals.

Reference(s):

1. Khakhum, N., et al., Burkholderia pseudomallei ΔtonB Δhcp1 Live Attenuated Vaccine Strain Elicits Full Protective Immunity against Aerosolized Melioidosis Infection. mSphere, 2019. 4: p. pii: e00570-18.
2. Hatcher CL, Mott TM, Muruato LA, Sbrana E, Torres AG.. Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders. Infect Immun, 2016. 84: p. 2345-2354.
3. Khakhum, N., et al., Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis. PLoS Negl Trop Dis, 2019. 13: p. e0007578.
4. Mott TM, Vijayakumar S, Sbrana E, Endsley JJ, Torres AG. Characterization of the Burkholderia mallei tonB Mutant and its potential as a backbone strain for vaccine development. PLoS Negl Trop Dis 2015. 9: p. e0003863.
5. Burtnick, M.N., et al., The cluster 1 type VI secretion system is a major virulence determinant in Burkholderia pseudomallei. Infect Immun, 2011. 79: p. 1512-1525.
6. Wiersinga, W., et al., Melioidosis. Nat Rev Dis Primers, 2018. 4: p. 17107.
7. Massey, S., et al., Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection. Sci Rep, 2014. 4: p. 4305.

Burkholderia pseudomallei Δasd strains (effective 12-13-2017)

The asd gene, encoding the aspartate semi-aldehyde dehydrogenase gene, is deleted in all B. pseudomallei strains listed in the table above. The asd mutants lack the ability to cross-link their peptidoglycan cell-wall and, in the absence of diaminopimelic acid (DAP), will die due to osmotic stress. DAP is a bacterial specific metabolite that is not made in hosts including mammals; therefore, these mutants are completely attenuated in vivo and are unable to replicate within the host.

Reference(s):

1. 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.
2. 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.

Burkholderia pseudomallei strain 576mn (effective August 18, 2017)
B. pseudomallei strain 576mn is a ∆purM derivative of the wild-type strain 576a. Studies demonstrated that strain 576mn was auxotrophic for adenine in minimal media, unable to replicate in human cells, significantly attenuated in BALB/C mice studies following high-dose intranasal inoculation (100% animal survival), and significantly attenuated compared to wild-type B. pseudomallei 576a strain.

Reference(s):

1. Norris MH, Rahman Khan MS, Schweizer HP, Tuanyok A. An avirulent Burkholderia pseudomallei ∆purM strain with atypical type B LPS: expansion of the toolkit for biosafe studies of melioidosis BMC Microbiol. 2017 Jun 7;17(1):132. doi: 10.1186/s12866-017-1040-4.

Burkholderia pseudomallei capsular polysaccharide mutant strain, JW270 (effective July 2, 2014)
The JW270 mutant contains a deletion of the capsule biosynthetic cluster (30.8 kb), a virulence determinant characterized in B. pseudomallei. Data from survival analysis and blood culture studies indicate significant attenuation (~4.46 log reduction) in hamster and murine models relative to wild-type B. pseudomallei strain DD503 (data not published).

Reference(s):

1. Atkins T, Prior R, Mack K, Russell P, Nelson M, Prior J, Ellis J, Oyston PC, Dougan G, Titball RW. Characterisation of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol. 2002 Jul;51(7):539-47.
2. Burtnick M, Bolton A, Brett P, Watanabe D, Woods D. Identification of the acid phosphatase (acpA) gene homologues in pathogenic and non-pathogenic Burkholderia spp. facilitates TnphoA mutagenesis. Microbiology. 2001 Jan;147(Pt 1):111-20.
3. Reckseidler SL, DeShazer D, Sokol PA, Woods DE. Detection of bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant. Infect Immun. 2001 Jan;69(1):34-44.
4. Reckseidler-Zenteno SL, DeVinney R, Woods DE. The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b deposition. Infect Immun. 2005 Feb;73(2):1106-15.
5. Reckseidler-Zenteno SL, Viteri DF, Moore R, Wong E, Tuanyok A, Woods DE. Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei. J Med Microbiol. 2010 Dec;59(Pt 12):1403-14. doi: 10.1099/jmm.0.022202-0. Epub 2010 Aug 19.
6. Sarkar-Tyson M, Thwaite JE, Harding SV, Smither SJ, Oyston PC, Atkins TP, Titball RW. Polysaccharides and virulence of Burkholderia pseudomallei. J Med Microbiol. 2007 Aug;56(Pt 8):1005-10.
7. Warawa JM, Long D, Rosenke R, Gardner D, Gherardini FC. Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis. Infect Immun. 2009 Dec;77(12):5252-61. doi: 10.1128/IAI.00824-09. Epub 2009 Sep 14.
8. Warawa JM, Long D, Rosenke R, Gardner D, Gherardini FC. Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant. Front Microbiol. 2011 Jun 16;2:133. doi: 10.3389/fmicb.2011.00133. eCollection 2011.

Burkholderia pseudomallei 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.

Reference(s):

1. 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.
2. 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.

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.

Reference(s):

1. 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 pseudomallei strain K96243 Δasd and MSHR487 Δasd (effective April 24, 2014)
Both strains contain a deletion in the aspartate-B-semialdehyde dehydrogenase (asd) gene, which is auxotrophic for diaminopimelate (DAP). In vivo, B. pseudomallei Δasd mutant strainsof K96243 and MSHR487 are attenuated with all mice surviving over a two month period after each animal was inoculated with 10⁷ CFUs (LD₅₀ for mice is approximately 10 CFU). No bacteria were recovered from the lung, liver, and spleen after challenging with a high dose of each mutant.

Reference(s):

1. 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.
2. 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.

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.

Reference(s):

1. 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.
2. 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.
3. 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.

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.

Reference(s):

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Rossi CA, Turell MJ. Characterization of attenuated strains of Rift Valley fever virus. J Gen Virol. 1988 Apr; 69(4):817-23.
9. 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.
10. 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.
11. 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.

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.

Reference(s):

1. 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.
2. 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.
3. 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.
4. Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC. Venezuelan equine encephalitis. Annu Rev Entomol. 2004; 49:141-74.
5. 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.
6. 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.
7. 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.
8. 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.

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.

Reference(s):

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

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).

Reference(s):

1. 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.

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.

Reference(s):

1. 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.

*Note: The modified Venezuelan Equine Encephalitis Virus TC-83(A3G) strain is a select agent and subject to select agent regulations (6, 7).

Reference(s):

1. 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
2. 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.
3. 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.
4. 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.
5. 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.
6. Hyde JL, Gardner CL, Kimura T, et al. A viral RNA structural element alters host recognition of nonself RNA. Science. 2014;343(6172):783-787. doi:10.1126/science.1248465
7. Kulasegaran-Shylini R, Thiviyanathan V, Gorenstein DG, Frolov I. The 5’UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins. Virology. 2009;387(1):211-221. doi:10.1016/j.virol.2009.02.027