Download Guidelines for Animal Research Involving Viral Vectors

GUIDELINES FOR ANIMAL RESEARCH INVOLVING VIRAL
VECTORS
General Containment
Commercial vector systems often come with a suggested biological safety containment level. A higher
containment level may be required depending on the specific properties of the vector and/or the insert of
interest. Additional safety considerations should be given to the design and use of viral vectors containing
potentially hazardous genes such as oncogenes, tumor suppressors, growth regulating products, etc. This
document provides general guidelines for animal biosafety containment for common viral vectors. The
biological safety level and containment requirements are ultimately determined by the Institutional Biosafety
Committee (IBC) and exceptions to these guidelines can be reviewed on a case by case basis.
ADENO-ASSOCIATED VIRUS VECTORS
Adeno-Associated virus (AAV) is a parvovirus infectious to humans and some other primate species. It is not known
to have any disease association, causes a mild immune response, and some AAV types are commonly found in the
general population. These viruses have the ability to integrate into a specific site on chromosome 19. Adenoassociated virus vector is replication incompetent and transduces dividing and non-dividing cells. It does not cause
disease. AVV vectors have also been rendered integration incompetent; the transferred DNA exists episomally. AAV
vectors have only a limited capacity to carry and transfer genes of interest. If the transferred gene does not encode
either a potentially tumorigenic gene product or toxin molecule, and the AAV vector is produced in the absence of a
helper virus, it can usually be handled at biosafety level 1.
Laboratory Hazards
•
•
Inhalation of aerosolized droplets, mucous membrane contact, parenteral injection, or ingestion.
There is no specific treatment for infection with AAV.
Animal Biosafety Containment
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•
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AAV vector can be administered under ABSL1 containment.
Animal housing maintained at ABSL1.
ABSL2 if helper virus is present.
Label cages with agent description. Biohazard sticker is not required.
Use microisolator top cages for 7 days after the final administration of viral vector.
Disposal and Decontamination
•
•
•
Autoclave bedding/cages if initial cage change or cage change is within 7 days post final administration prior
to disposal or washing.
Dispose of carcasses as pathology waste.
Use 10% household bleach solution or other appropriate EPA approved disinfectant, or autoclave for 30
minutes at 121ºC under 15 lbs per square inch of steam pressure.
ADENOVIRUS VECTORS
Adenoviruses are infectious to humans and animals and often cause mild respiratory illness, pink eye or
gastroenteritis (stomach flu). In rare cases, particularly in immunocompromised individuals, severe disease can
occur. Adenovirus and adenoviral vectors can infect a wide range of mammalian cells and replicate as episomal
elements in the nucleus of the host cell, not integrating into the host genome. The vector is recombinogenic if wild
type adenovirus is somehow introduced into producer cell culture and can become replication competent.
Laboratory Hazards
•
•
Inhalation of aerosolized droplets, mucous membrane contact, parenteral injection, or ingestion.
Adenovirus is stable in the environment.
Animal Biosafety Containment
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•
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Adenovirus vector must be administered under ABSL2 containment with use of a biosafety cabinet.
Animals may shed/excrete adenovirus post-administration. Animals must be housed under ABSL2
conditions for 7 days post final administration, after which animals may be housed at ABSL1.
Label cages with agent description. Biohazard sticker is required.
Use microisolator top cages for 7 days after the final administration of viral vector.
Disposal and Decontamination
•
Autoclave bedding/cages if initial cage change or cage change is within 7 days post final administration prior
to disposal or washing.
•
•
Dispose of carcasses as pathology waste.
Use 10% household bleach solution or other appropriate EPA approved disinfectant, or autoclave for 30
minutes at 121ºC under 15 lbs per square inch of steam pressure.
RETROVIRUS/MURINE LEUKEMIA VIRUS (MLV) VECTORS
Moloney Murine Leukemia Virus, short MMLV or MLV, is a retrovirus that infects actively diving cells and integrates
in the host genome. While the wild-type virus is oncogenic in mice, it is not known to cause clinical manifestations of
disease in humans. The host range of MLV or MMLV vectors is dependent on the specificity of the viral envelope.
Pseudotyping directs tropism (e.g. ecotropic – infects mouse cells, amphotropic – infects human and mouse cells,
VSV-G – vesicular stomatitis virus G protein - infects all mammalian and other vertebrate cells = pantropic). MLV
vectors have a large gene carrying capacity. They only transduce dividing cells, which can be seen as an advantage
or disadvantage for certain applications. MLV vectors integrate semi randomly into start regions of active genes in
the host chromosome, which poses a risk of insertional mutagenesis and oncogenesis. Gene expression by MLVbased vectors can be shut down in human cells over time.
Laboratory Hazards
•
•
In mice, virus is transmitted via blood from infected mother to offspring; may also occur via germline
infection.
Infection in humans appears to require direct parenteral injection with amphotropic or pseudotyped MLV.
Animal Biosafety Containment
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Ecotropic MLV vector can be administered under ABSL1 containment.
Amphotropic or pseudotyped MLV vector must be administered under ABSL2 containment with use of a
biosafety cabinet.
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If using amphotropic or pseudotyped MLV vector, animals must be housed under ABSL2 conditions for 48
hours post final administration, after which animals may be housed at ABSL1.
Label cages with agent description. Biohazard sticker is required for amphotropic or pseudotyped MLV.
Use microisolator top cages for 48 hours after the final administration of viral vector.
Disposal and Decontamination
•
•
•
Autoclave bedding/cages if initial cage change or cage change is within 48 hours post final administration
prior to disposal or washing.
Dispose of carcasses as pathology waste.
Use 10% household bleach solution or other appropriate EPA approved disinfectant, or autoclave for 30
minutes at 121ºC under 15 lbs per square inch of steam pressure.
LENTIVIRUS VECTORS
Lentiviruses are a subset of retroviruses. HIV-1 is the best known example of this subset of retroviridae and is the
cause of AIDS in humans. They integrate into active genes in the host cell chromosomes, but not into transcriptional
start regions. They can infect dividing and non-dividing cells. They have the potential of causing insertional
mutagenesis. They express their genes in a stable fashion long term. Just as the wild type viruses, lentiviral vectors
transduce both dividing and non-dividing cells. They semi-randomly integrate into the host genome, but their
integration patterns are much safer than the integration patterns of MLV vectors, since they do not integrate into
transcriptional start regions. Therefore, activation of oncogenes in cis is less prevalent. They can still cause
insertional mutagenesis, however. They have a large gene carrying capacity and provide stable, long term gene
expression in human cells. Lentivectors are mainly pseudotyped with VSV-G, which makes them pantropic.
Lentivectors are mainly pseudotyped with VSV-G, which makes them pantropic. Production systems for lentivectors
are commercially available in 3 or 4 plasmid systems to lower the risk for recombination and production of
replication-competent lentivectors in the producer cells. However, generation of replication-competent lentivector
cannot be completely excluded and needs to be verified. It has, however, never occurred since the inception of
these systems. Post Exposure Prophylaxis with antiretroviral drugs can be considered if there has been an
exposure incident with lentiviral vectors under the direction of the Occupational Health Physician.
Laboratory Hazards
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Direct contact with skin and mucous membranes, parenteral injection, or ingestion.
Animal Biosafety Containment
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•
•
•
Lentivirus vector must be administered under ABSL2 containment with use of a biosafety cabinet. Additional
containment requirements may be necessary depending on the gene insert.
Animals must be housed under ABSL2 conditions for 48 hours post final administration, after which animals
may be housed at ABSL1.
Label cages with agent description. Biohazard sticker is required.
Use microisolator top cages for 48 hours after the final administration of viral vector.
Disposal and Decontamination
•
•
•
Autoclave bedding/cages if initial cage change or cage change is within 48 hours post final administration
prior to disposal or washing.
Dispose of carcasses as pathology waste.
Use 10% household bleach solution or other appropriate EPA approved disinfectant, or autoclave for 30
minutes at 121ºC under 15 lbs per square inch of steam pressure.
Viral Vector
AdenoAssociated
Virus (AAV)
Risk
Group
Animal
Biosafety
Level
Special Handling Requirements ***
Hold
Caging during Labeling
Period**
hold period
Label with
description of
agent and dose
1
ABSL1
7 days
Microisolator lid
Biohazard
sticker not
required
Adenovirus
2
ABSL2
7 days
Microisolator lid
Label with
description of
agent and dose
Biohazard
sticker required
Retrovirus/
Murine
Leukemia
Virus (MLV)
Label with
description of
agent and dose
1 / 2*
ABSL1 /
ABSL2*
48 hours
Microisolator lid
Lentivirus
2
ABSL2
48 hours
Microisolator lid
Biohazard
sticker required
for amphotropic
or pseudotyped
Label with
description of
agent and dose
Biohazard
sticker required
Carcass
Disposal
Biohazard
(Pathology)
Waste
Biohazard
(Pathology)
Waste
Bedding/Cage
Disposal ****
Autoclave bedding
and cages prior to
disposal/washing
during hold period
After hold, discard as
standard waste
Autoclave bedding
and cages prior to
disposal/washing
during hold period
After hold, discard as
standard waste
Biohazard
(Pathology)
Waste
Autoclave bedding
and cages prior to
disposal/washing
during hold period
After hold, discard as
standard waste
Biohazard
(Pathology)
Waste
Autoclave bedding
and cages prior to
disposal/washing
during hold period
After hold, discard as
standard waste
*If amphotropic or pseudotyped
** Holding time that must be observed before animals can be downgraded to ABSL1 housing
*** Special handling requirements for non-rodents (for example: livestock and primates) may vary or differ from these guidelines. Specific procedures must
be approved by the IBC.
**** Autoclave initial cage change regardless of hold period