Environmental Health and Safety

Viral Vector BSDS

Adeno-Associated Virus (AAV)

Types 12 serotypes (AAV2 tropism: muscles, neurons, hepatocytes)
NIH Risk Group 1 (AAV 1-4); unless oncogene/toxin or helper virus present (RG-2)
Biocontainment Level BSL-1; unless oncogene/toxin or helper virus present (BSL-2)
Infectious to Humans/Animals Yes (Humans/Primates)
Route of Transmission:  Inhalation
Disease: Not known to cause direct disease in humans; causes mild immune response
Treatment: Supportive care
Safety Considerations Infects multiple cell types. May be associated with insertional mutagenesis and cancer. Inserts itself on human chromosome 19 and remains latent. Can be potentially reactivated later in the presence of a helper virus and produce infection.
Integration: Selective
Replication Compentent: No — needs helper virus (CMV, adenovirus, herpesvirus, vaccinia)
RCV Testing: If helper virus is adenovirus, presence of RCV after heat inactivation (56C for 15min)
Recombination: Possible. Use of 293 cells with E1 deletion can cause systematic rescue
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic:
Safe Use 1) Gutless systems (Cre-loxP)
2) 2nd Generation: Deletion of regions E1, E2, E3, E4 genes
3) 1st Generation: Deletion of regions E1, E3 genes (less safe)
Disinfection: 10% bleach (fresh)
Animals ABSL-1; ABSL-2 in the presence of a helper virus or if host animal could house helper virus. Special handling of bedding and cages for 48 hours post injection. Bedding disposed in biohazardous waste.
Vendors Vector Biolabs, Cell Biolabs

Adenovirus

Types Ad 1, 2, 3, 4, 5, 7, 40, 41
NIH Risk Group 2
Biocontainment Level BSL-2. No open bench work. Use eye protection. Stable in environment
Infectious to Humans/Animals Yes
Route of Transmission:  Oral/Inhalation
Disease: Primarily upper respiratory. Generally mild. Can cause eye damage.
Treatment: Supportive care
Safety Considerations Can infect a variety of non-dividing cells. Cytotoxic (T-cell response)
Integration: No
Replication Competent: Possible
RCV Testing: Recommended- PCR for E1 prior to use; plate on E1 non-susceptible cell types
Recombination: Possible. Use of 293 cells with E1 deletion can cause systematic rescue of wild type
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic:
Safe Use 1) Gutless systems (Cre-loxP
2) 2nd Generation: Deletion of regions E1, E2, E3, E4 genes
3) 1st Generation: Deletion of regions E1, E3 genes (less safe)
Disinfection: 10% bleach (fresh); Do not use 70% alcohol.
Animals ABSL-2 housing for 7 days post-injection (replication incompetent); ABSL-2 for xenografts of human/animal cells infected. During post infection period: Animals manipulated in BSC. Microisolators used. Adenovirus is excreted by animals. Cage changes/dumping in BSC. Spray cages/racks with virucide.
Vendors Agilent (AdEasy), MP Biomedicals (AdenoVator), Invitrogen (ViraPower)

Baculovirus

Types Autographa californica
NIH Risk Group 1
Biocontainment Level BSL-1
Infectious to Humans/Animals Possible with mammalian promoter
Route of Transmission: Direct contact
Disease: No known disease
Treatment: NA
Safety Considerations Can be modified to infect mammalian cells.
Integration: Yes
Replication Competent: No
Recombination:
Pseudotyped: Possible
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic: Evaluate
Safe Use Disinfection: 10% bleach (fresh); 70% ETOH
Animals ABSL-1
Vendors  

Retrovirus (Lentivirus)

Types Human (HIV 1-3), Human T-cell Lymphotropic Virus Type 4 (HTLV), Simian (SIV), Feline (FIV)
NIH Risk Group 2 or 3 (HIV)
Biocontainment Level Nonhuman (BSL-2), Human (BSL-2+ or higher)
Infectious to Humans/Animals Yes
Route of Transmission: Bloodborne
Disease: Can cause severe immunologic and neurological disease in hosts
Treatment: Available for HIV (NRT inhibitors, Protease inhibitors, others)
Safety Considerations Insertational mutagenesis. Can infect non-dividing cells including immune cells. Can infect non-target cells. Can persist lifelong. High mutation rates. Inappropriate expression of gene product. Rescue by other human pathogenic viruses
Integration: Yes
Replication Competent: Possible
RCV Testing: Serial transfer and ELISA assay for P24 antigen at 1 infectious unit/mL
Recombination: Possible. (Decreases with later generations) Use transient production system
Pseudotyped: VSV-G (extends host tropism)
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic: Varies
Safe Use 1) Use third generation system: recommend multiple plasmid system with Gag, Pol, Env genes on separate plasmids.  Risk decreases from two plasmids to four (recombination/replication competent)
2) Tat gene deleted (replication)
3) Use non-native Env gene. Use non-primate vectors if possible. 
Disinfection: 10% bleach (fresh)
Animals Is host permissive?
ABSL-2 housing for 48 hours post-injection (replication incompetent); ABSL-2 for xenografts of human/animal cells infected. Safe needle devices for injection.
Vendors Invitrogen

Murine Retroviruses

Types Moloney Murine Leukemia Virus: (MoMuLV), Mouse Mammary Tumor Virus (MMTV)
NIH Risk Group 1 (if ecotropic); 2 for others
Biocontainment Level BSL-1 if ecotropic and replication incompetent; BSL-2 for others
Infectious to Humans/Animals Humans: possible if pseudotyped
Route of Transmission: Bloodborne
Disease: Cell transformation and tumor formation
Treatment:
Safety Considerations Insertational mutagenesis possible, leading to cell transformation/tumor formation. Amphotropic Env gene can infect non-murine cells including human cells
Integration: Yes
Replication Competent: Possible
RCV Testing: Use permissive cell line (Mus dunni); screen by marker rescue assay (PG-4S+L-)
Recombination: Possible (Helper virus or endogenous virus in cell lines can increase risk)
Pseudotyped: Yes
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic: Varies based on packaging cell line
Safe Use 1) Use ecotropic Env gene
Disinfection: 10% bleach (fresh)
Animals Depends on construct. ABSL-1 for ecotropic. Virus will be shed in bedding. ABSL-2 if infects humans.
Vendors Invitrogen

Sindbis (alphavirus/arbovirus)

Types  
NIH Risk Group 2
Biocontainment Level BSL-2 (Stable up to two days)
Infectious to Humans/Animals Yes (insects/birds/rodents/humans)
Route of Transmission: Bloodborne
Disease: Self limiting febrile disease, rash, arthritis
Treatment:
Safety Considerations Cytotoxic: virus can induce apoptosis
Integration:
Replication Competent:
RCV Testing:
Recombination:
Pseudotyped:
Transgene: Evaluate if oncogenic or biotoxic
Ecotropic/Xenotropic/Amphotropic:
Safe Use Disinfection: 10% bleach (fresh)
Animals ABSL-2
Vendors