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Biosafety & Regulations Varsha 2024 19-08-2024 150 | H A Z A RD ASSE SSM E NT Figure 1: Spore formation provides an efficient mechanism for dispersal of molds. Spores from some species pose a risk of infection to laboratory personnel. In this image of Coccidioides immitis, cells of a ve...

Biosafety & Regulations Varsha 2024 19-08-2024 150 | H A Z A RD ASSE SSM E NT Figure 1: Spore formation provides an efficient mechanism for dispersal of molds. Spores from some species pose a risk of infection to laboratory personnel. In this image of Coccidioides immitis, cells of a vegetative hypha have transformed into spores (arrows) that will be liberated when walls of the adja- cent cells, now dead and withered, become fractured (arrow- heads). A recent survey found that laboratory compliance.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.co Laboratory-acquired mycoses Fungus BSL/ABSL Route Infectious potential No. cases References Blastomyces dermatitidis 2/2 Transcutaneous Serious/possibly fatal 12 124 125 17 126 62 Inhalation Serious /possibly fatal 2 62 64 Candida albicans 2?a/2?a Cutaneous Minor 1b 28 Coccidioides posadasiic 3d/2 Inhalation Serious/possibly fatal Numerouse 17 Transcutaneous Serious/possibly fatal 4 127 Spray Serious 1 69 Coccidioides immitisc 3d/2 Inhalation Serious/possibly fatal Numerousc 17, 128, 129 Cryptococcus neoformans 2/2f Transcutaneous Serious 1 35 Dermatophytes 2/2 Cutaneous Minor Numerous 82 17 81 Penicillium marneffei g 2/2 Transcutaneous Serious 1 100 Inhalation (presumed) Serious/possibly fatal 1 101 Histoplasma capsulatum 3d/2 Inhalation Serious/possibly fatal Numerous 17 Transcutaneous Serious 3 88 89 90 Spray Serious 1 91 Sporothrix schenckii 2/2 Transcutaneous Serious ca. 2 dozenh 17 105 Spray Serious 3 17 130 a See discussion under “Considerations for Specific Fungi” (subhead “Yeasts”), below.  TABLE 1. TABLE 1. y.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative ey.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons a Summary of previously published laboratory-associated viral infections (Continued) No. of No. of Family Genus Virus cases No. of deaths Recommended biosafety levelb Family Genus Virus cases No. of deaths Recommended biosafety levelb Adenoviridae Aviadenovirus Fowl plague virus 1 0 2 Louping-ill virus 67 0 3 Mastadenovirus Adenovirus 10 0 2 Omsk hemorrhagic fever virus 9 0 4 Arenaviridae Arenavirus Lassa virus 3 0 4 Powassan virus 2 0 3 Lymphocytic choriomeningitis 102 9 2 for work with infectious Rio Bravo virus 12 0 2 (LCM) virus material or infected animal, 3 St. Louis encephalitis virus 3 0 3 for activities with high potential Spondweni virus 4 0 3 for aerosol production or production of large quantities Tick-borne encephalitis (TBE) 39 2 3 of material, or work with virus infected hamsters Wesselsbron virus 10 0 3 Junin virus 15 1 4 (level 3 if immunized) West Nile virus 27 0 3 Machupo virus 7 1 4 Yellow fever (YF) virus 140 24 3 Sabia virus 2 0 4 Zika virus 4 0 2 SPH114202 virus 1 0 4 Negishi virus 1 0 3 Bunyaviridae Nairovirus Crimean-Congo hemorrhagic 5 0 4 Hepadnaviridae Orthohepadnavirus Hepatitis B virus (HBV) 19 0 2; 3 for activities with potential for droplet or fever virus (CCHFV) aerosol production and viral Dugbe virus 1 0 3 concentration Nairobi sheep disease virus 3 0 3 Herpesviridae Lymphocryptovirus Epstein-Barr virus 2 0 2; 3 for producing, purifying, Orthobunyavirus Apeu virus 2 0 2 and concentrating Marituba virus 2 0 2 Simplexvirus Herpes B virus 37 20 2 for tissue specimens from macaques; 3 for suspected Oriboca virus 2 0 2 infected tissue or in vitro Bunyamwera virus 8 0 2 propagation, 4 for cultures with Germiston virus 5 0 3 high-titer virus Oropouche virus 5 0 3 Herpes simplex virus 2 0 2; 3 for producing, purifying, and concentrating Guaroa virus 1 0 2 Varicellovirus Pseudorabies virus 2 0 2 Ossa virus 1 0 2 Bovine encephalomyelitis virus 1 0 Unclassified Phlebovirus Rift Valley fever (RVF) virus 103 4 3 Varicella-zoster virus 3 0 2 Hantavirus Hantaan virus 226 0 2 for infected tissue samples Orthomyxoviridae Unspecified Influenza virus 22 1 2; 3 for highly pathogenic using level 3 practices, 3 for avian influenza viruses (HPAI) cell culture propagation, 4 for and the 1918 influenza virus large-scale growth and viral strain concentration Paramyxoviridae Rubulavirus Newcastle disease virus 77 0 Restricted animal pathogen Coronoviridae Coronavirus Middle East respiratory 0 0 2; 3 for cell culture propagation syndrome coronavirus Mumps virus 8 0 2 (MERS-CoV) Morbillivirus Measles virus 2 0 2 Severe acute respiratory 6 0; but a 2; 3 for cell culture propagation Pneumovirus Respiratory syncytial virus 1 0 2 syndrome coronavirus researcher in (RSV) (SARS-CoV) China subse- Respirovirus Sendai virus 1 0 2 quently infected Parvoviridae Erythrovirus B19 virus 10 0 2 her mother, who Picornaviridae Aphthovirus Foot-and-mouth disease virus 2 0 Restricted animal pathogen succumbed to the infection Cariovirus Mengovirus 2 0 encephalomyocarditis Filoviridae Marburgvirus Marburg virus 31 9 4 Enterovirus Coxsackie virus 39 0 Ebolavirus Ebola virus 4 1 4 Poliomyelitis virus 21 5 2 Flaviviridae Flavivirus Dengue virus 14 0 2 Swine vesicular disease 1 0 Restricted animal pathogen Japanese encephalitis virus 2 0 3 Echovirus 3 0 2 Kunjin virus 4 0 2 Hepatovirus Hepatitis A virus (HAV) 5 0 2 Kyasanur Forest disease (KFD) 132 0 4 virus (continued )  m/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; O (Continued) No. of Family Genus Virus cases No. of deaths Recommended biosafety levelb Poxviridae Orthopoxvirus Vaccinia and smallpox viruses 38 0 2 for facilities with three practices (if immunized, smallpox an exception; work on live variola can only be conducted within two approved BSL4/ABSL4 facilities: CDC, Atlanta, GA, and the State Research Center of Virology and Biotechnology,VECTOR, in Koltsovo, Russia) Yatapoxvirus Yaba and Tana viruses 24 0 2 (if immunized) Parapoxvirus Orf virus 2 0 2 Reovirus Coltivirus Colorado tick fever virus 19 0 2 Retroviridae Lentivirus Human immunodeficiency 45 0 2; 3 for industrial scale or high virus (HIV) concentrations of virus Simian immunodeficiency virus 3 0 2; 3 for industrial scale or high (SIV) concentrations of virus Oncornavirinae Simian type D retrovirus 2 0 Spumavirus Simian foamy virus 13 0 2 Rhabdoviridae Lyssavirus Rabies virus 2 0 2; 3 for aerosol-producing activities or high concentrations of virus Vesiculovirus Vesicular stomatitis virus 78 0 3 (exception for laboratory- adapted strains, level 2) Piry virus 10 0 3 Togaviridae Alphavirus Chikungunya virus 33 0 3 Eastern equine encephalitis 7 0 2 (level 3 and immunized if virus working with infection of newly hatched chickens) Mayaro virus 6 0 3 Mucambo virus 4 0 3 Venezuelan equine encephalitis 187 2 3 (VEE) virus Western equine encephalitis 16 4 2 (level 3 if immunized and (WEE) virus working with infection of newly hatched chickens) Rubivirus Rubella virus 7 0 Unspecified Unspecified Hepatitis virus 360 2 2 Unspecified Unspecified Viral diarrhea virus 2 0 Unspecified Unspecified Hemorrhagic nephrosonephritis 1 0 virus Data from references 6–9, 46, 66, 81, 82, 126, 157, 212–214, 227–253. a 94 | HAZARD ASSESSMENT RISK MATRIX Risk is a function of likelihood and consequences. To b able to adequately assess risk, laboratorians must unde stand the underlying hazard posed by live viral agents the laboratory environment as well as the consequence infection with various viruses. Figure 1 provides a pote tial risk matrix that can be used by the laboratorian assess risk. The hazard probability is an estimate base on the type of procedures used in the laboratory and th  Downloaded from https://onlinelibrary.wiley.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2 VIRAL VECTOR TECHNOLOGY re pro duc ing Figure 1: Number of PubMed citations found for recombinant viral vectors. Figure 1: Number of PubMed citations found for recombinant viral vectors. Downloaded from https://onlinelibrary.wiley.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/202 T I FI CAT I O N VIRAL VECTOR TECHNOLOGY reproducing and disseminating their nucleic ZARD IDENTIFICATION Viruses are desirable systems for recombinant gene de- sponse load VIRAL VECTOR TECHNOLOGYreprothrough ducingout to that andload a pop dis spread, ulation sem i is natthrough of cells, ing mainly load throughout a population of cells, their and nu respon the clei andsith b Viruses are desirable systemslivanderyfor livery plat Viruses are deplat liver re forms com desirdue abletosys nucleic forms due ac toids tobi their tems their abil for nant ityre newity abil tocom conbi cells. to conThis gene dense, nant pack prop dense, of de- geneage, de- path ertyage, pack ogenic sponse ex ploit path ogethe ity ofsponse to ity of aspread, that nicgene de viral infec a liv is tion mainly eryincafec viral pation to in a re bilityinof host. sponTh a ahost. t si virus, T livery platforms due to their ability to condense, package, pathogenicit and deliver nucleic acids to new cells. This property of exploit the gene delivery capability of a viru and deliver nucleic acids to new cells. This property of exploit the g Figure 2: Number of human gene therapy clinical protocols approved worldwide by year as of July 2015. Source da reference 182. Figure 1: Figureof Number Number of 1:PubMed PubMed cita citations tions found for found recomfor recom binant vibi ralnant vecvitors. ral vec tors. 2: Number of human gene therapy clinical protocols approved worldwide by year as of July 2015. Source d Figure reference 182.  com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.wile Viral vectors and transgene containment Laboratory containment Gene transfer vectora Host rangeb Insert or gene functionc leveld MMLV based, gag, pol, env deleted Ecotropic S, E, M, G, CC, T, MP, DR, R, TX, Ov, Oc, BSL1* Amphotropic, VSV-G pseudotyped S, E, M, MP, DR, T, G, BSL2 Ov, Oc, R, CC BSL2+ TX BSL3 Herpes virus based, nonlytic Broad host range, nervous system S, E, M, MP, DR, T, G BSL2 Ov, Oc, R, CC BSL2+ TX BSL3 Lentivirus based, HIV, SIV, EIAV, FIV, Ecotropic, amphotropic, VSV-G S, E, M, MP, DR, T, G BSL2 etc.; gag, pol, env, nef, vpr deleted pseudotyped Oc, Ov, R, CC BSL2+ TX BSL3 Adenovirus based, serotypes 2, 5, Broad host range, infective for many S, E, M, T, MP, DR, R, G, CC BSL2 7; E1 and E3 or E4 deleted cell types Ov, Oc, BSL2+ TX BSL3 Rabies virus, SAD B19 strain Broad mammalian host range, nervous Marker proteins such as EGFP BSL2 (veterinary vaccine strain), G protein system specific Ov, Oc, BSL2+ deleted TX BSL3 Baculovirus based Broad mammalian host cell range S, E, M, T, MP, DR, R, G, CC BSL1* Ov, Oc, BSL2 TX BSL2+/BSL3 AAV based, rep, cap defective Broad host range, infective for many S, E, M, T, MP, DR, G BSL2 cell types including neurons Ov, Oc, R, CC BSL2+ TX BSL2+/BSL3 Poxvirus based, canarypox, vacciniae Broad host range S, E, M, T, DR, MP, CC, R, G, BSL2 Ov, Oc, BSL2+ TX BSL3 a  AAV based, rep, cap defective Broad host range, infective for many S, E, M, T, MP, DR, G BSL2 cell types including neurons Ov, Oc, R, CC BSL2+ TX BSL2+/BSL3 Poxvirus based, canarypox, vacciniae Broad host range S, E, M, T, DR, MP, CC, R, G, BSL2 Ov, Oc, BSL2+ TX BSL3 a Refers to the parental or wild type virus and some of the common deletions used in viral vectors. b Refers to ability of vector to infect cells from a range of species. Ecotropic generally means able to infect only cells of species originally isolated from or identified in. Please note that the ecotropic host for HIV and HSV would be human cells, but the ecotropic host for MMLV would be murine cells. Amphotropic and VSV-G pseudotyped virus host range includes human cells. c These are general categories of marker or cellular genes and functions. Please note that there are differences in the containment level for the same gene class, depending on whether the viral vector integrates into the recipient genome at high rate. The general categories are: EGFP, enhanced green fluorescent protein; S, structural proteins, actin, myosin, etc.; E, enzymatic proteins, serum proteases, transferases, oxidases, phosphatases etc.; M, metabolic enzymes, amino acid me- tabolism, nucleotide synthesis, etc.; G, cell growth, housekeeping; CC, cell cycle, cell division; DR, DNA replication, chromosome segregation, mitosis, meiosis; MP, membrane proteins, ion channels, G-coupled protein receptors, transporters, etc.; T, tracking genes such as GFP, luciferases, photoreactive genes; TX, active subunit genes for toxins such as ricin, botulinum toxin, Shiga and Shiga-like toxins; R, regulatory genes, transcription and cell activators such as cytokines, lymphokines, tumor suppressors; Ov and Oc, oncogenes identified via transforming potential of viral and cellular analogs, or mutations in tumor suppressor genes resulting in a protein that inhibits/moderates the normal cellular wild-type protein. This does not include SV40 T antigen. SV40 T antigen-containing cells should not be considered more hazardous that the intact virus. SV40 is considered a Risk Level 1 agent (the lowest level) according to the NIH Guidelines (Appendix B in reference 57). The prevalence of SV40 infection in the U.S. population due to contaminated polio vaccine does not seem to have caused a statistically significant increase in the rate of cancers. d This is a general assessment of containment levels for laboratory construction and use of these vectors for nonproduction quantities only based on the 2009 edi- tion of BMBL (101). Please note this table cannot cover every potential use within a research or laboratory setting, as information is gained risk assessments and containment levels may be changed. Local IBCs should use all available information and their best judgment to determine appropriate containment levels. BSL1* refers to the containment level based on parent virus risk group; however, most procedures involving the handling and manipulation of the viral vectors are done at BSL2 to protect cell cultures and viral stocks from contamination. e Certain specific strains of poxviruses, such as MVA, NYVAC, ALVAC, and TROVAC, are considered low-risk agents and can be handled at BSL1 in certain cases (see “Poxvirus Vectors” section). A toxin is defined as “a poisonous substance that is a specific product of the metabolic activities of a living organism and is usually very unstable, notably toxic when introduced into the tissues, and typically capable of inducing antibody formation”  TABLE 1. ley.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlin Sources and mechanisms for various toxins and venoms (16) Toxin or class Source(s) Mechanism of action Small molecules Tetrodotoxin Puffer fish, octopus, salamander Na+ channel blocker Saxitoxin Shellfish contaminated with dinoflagellates Na+ channel blocker Ciguatoxin Large tropical fish contaminated with dinoflagellates Actions on Na+ channel Cardiac glycosides Toad skin Adenosine triphosphatase (ATPase) inhibitor Batrachotoxin Frog skin Central ner vous system toxin Palytoxin Sea anemone Lanophore Proteins and polypeptides α-Bungarotoxin Elapid snakes (kraits) Nicotinic receptor blocker β-Bungarotoxin Elapid snakes (kraits) Presynaptic cholinergic nerves α-Conotoxin Cone shells Nicotinic acetylcholine receptor ligand μ-Conotoxin Cone shells Skeletal muscle Na+ channel blocker w-Conotoxin Cone shells N-type Ca2+ antagonist Cardiotoxin Elapid snakes Direct-acting cardiotoxin Phospholipases Many snakes Cell membrane destruction Bacterial toxins Botulinum toxin Clostridium botulinum Synaptin in nerve endings Cholera toxin Vibrio cholerae Activation of Gs protein Pertussis toxin Bordetella pertussis Inactivates Go /Gs protein Endotoxin Gram-negative bacteria Cell membranes Tetanus toxin Clostridium tetani Cell membrane ionophore Staphylococcal toxin Staphylococcus spp. Enterotoxin Pseudomonas exotoxin A Pseudomonas aeruginosa Inhibits protein synthesis Diphtheria toxin Corynebacterium diphtheriae Adenosine diphosphate (ADP)-ribosylation of elongation factor 2  ed from https://onlinelibrary.wiley.com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024 Bacterial toxins are “soluble substances that alter the normal metabolism of C H A P T E R 11 : B I O LO G I CAL TOXI N S: SAF ETY AN D SCI EN CE | 251 host cells with deleterious effects on the host” and are the primary virulence factors TABLEfor 3. a variety of pathogenic bacteria. Primary features of bacterial exotoxins and endotoxins (14) Exotoxins Endotoxins Excreted by living cells; high concentrations in liquid medium Integral part of the cell wall of Gram-negative bacteria; released during bacterial death and in part during growth; may not need to be released to have biological activity Produced by both Gram-positive and Gram-negative bacteria Produced only by Gram-negative bacteria Polypeptides Lipopolysaccharide complexes Relatively unstable; toxicity often destroyed rapidly at Relatively stable; withstand heating at temperatures above 60°C temperatures above 60°C for hours without loss of activity Highly antigenic; stimulate formation of high-titer antibodies Weakly immunogenic; relationship between antibody titers and protection from disease is less clear than with exotoxins Converted to antigenic toxoids when inactivated by treatment Not converted to toxoids with formalin, acid, heat, etc. Toxoids are used for preventive immunization (i.e., tetanus toxoid) Highly toxic; fatal to animals in a dose of microgram Moderately toxic; fatal to animals in a dose of tens to hundreds of quantities or less micrograms Usually bind to specific receptors on cells Specific receptors not found on cells Usually do not produce fever in the host Usually produce fever in the host through the release of interleukin-1 and other mediators Frequently controlled by extrachromosomal genes (i.e., plasmids) Synthesis directed by chromosomal genes  com/doi/ by Indian Institute Of Science Education And Research Thiruvananthapuram (IISER TVM), Wiley Online Library on [28/07/2024]. See the Terms and Conditions (https://onlinelibrary.w Toxicity data for Select Toxins Toxic dosea Toxin type Toxin source Toxin name Mice Guinea pigs Rabbits Monkeys Humans Bacterial toxins Bacillus anthracis LFPA 1.25 μg i.v. Clostridium botulinum 2.4 μg i.v. (rat) Type A Neurotoxin A 1.2 ng i.p. (0.6 ng)b (0.5 ng)b (0.5–0.7 ng)b (ca. 1 ng)b Type B Neurotoxin B 0.5 ng i.v. 0.6 ng i.p. (proteolytically activated) Type C Neurotoxin C1 1.1 ng i.v. (ca. 1.1 ng)b (ca. (ca. 0.4 ng)b 0.15ng)b Type D Neurotoxin D 0.4 ng 0.1 ngb 0.08 ngb 40 ngb Type E Neurotoxin E (1.1 ng) 0.6 ngb 1.1 ngb 1.1 ngb Type F Neurotoxin F 2.5 ng i.p. Clostridium tetani Tetanus toxin (0.1 ng) (ca. 0.3 ng)b (0.5–5 ng)b (