This training module is required for all personnel listed on an IBC protocol that describes work utilizing viral vectors (both replication‐competent and‐incompetent) regardless of the biosafety level used to manage them. Each individual shall complete this course every five years unless a significant change in legal or institutional policy or safety guidelines dictate a shorter time interval.
1 2 3 4 5 Pathogenicity of viruses is generally linked to their ability to replicate and disseminate in host cells.
In order to address this, replication‐competent vectors are usually disabled or attenuated in some way: ‐ Deletion of genes encoding structural proteins ‐ Replication competence in only a particular species
6 7 The genetic material to be packaged into the viral vector is introduced into a packaging or helper cell. This genetic material will not encode the viral components required to replicate and package a virus but will contain packaging signals for incorporation into a virion. The packaging or helper cell will contain the genetic material required to create virions (such as structural proteins). This genetic material is supplied via transfection or from a packaging cell line that has had these genes stably incorporated into the cellular genome. The packaging or helper cell will then provide the viral packaging components in trans, producing virions that have packaged the vector genetic material. These virions can be collected and used to infect a target cell. The virion will enter the target cell, but because the viral genes (previously provided by the packaging or helper cell) are not present, no viral progeny are produced. In some cases, helper plasmids or helper viruses may be used in lieu of helper cells.
8 Visit the University of Chicago Institutional Biosafety Committee (IBC) for further information on testing requirements for viral vectors http://researchadmin.uchicago.edu/docs/ibc/ibc_Testing_Requirements_Viral_Vectors.pdf
9 10 11 Baculovirus has been shown to enter mammalian cells and deliver nucleic acid, although it cannot replicate and produce progeny virions in these cells. If the recombinant nucleic acid is engineered to have a mammalian promoter, the genes will be expressed in mammalian cells. Special consideration must be given to the nature of the recombinant nucleic acid expressed in mammalian cells. For example, if a baculoviral vector is engineered to deliver an oncogene under the control of a mammalian promoter, a risk assessment must be performed (by the investigator and the Institutional Biosafety Committee via submission of an IBC protocol, required for all research involving rDNA) to consider the enhancement of the biosafety level used to mitigate biosafety risks.
12 For BSL‐1 and BSL‐2 laboratories, the NIH Guidelines stipulates that Appendix G‐II‐A‐1‐c and Appendix G‐II‐B‐1‐c: All contaminated liquid or solid wastes are decontaminated before disposal.
13 14 15 Ability of retroviruses to transduce cells of different species is controlled by the sequence of the env gene and the cell surface receptor it recognizes. The tropism of a virus can be modulated by pseudotyping.
Ecotropic: viruses that are only able to infect the species from which originally isolated
Xenotropic: viruses that are unable to infect the species from which originally isolated
Amphotropic: viruses that are able to infect multiple species of mammalian cells, including human
Pantropic: viruses that are able to infect both mammalian and non‐mammalian cells, including human
16 Animal Biosafety Level (ABSL)
17 IBC Testing Requirements for Viral Vectors
Viral preparations for in vitro experiments of short duration (used within 72 hours of viral particle generation): Every six months, a representative viral preparation must be tested to ensure RCVs are not being produced.
Every producer cell line or stably transduced cell line generated or placed in a laboratory repository must be tested for RCV prior to use.
If a viral preparation is being administered to animals directly, it must be tested for RCV. If cells are being transduced with the intent of administering the cells to animals, you must either test the viral preparation for RCV before transduction or test the cells for RCV following transduction.
Marker rescue, antibiotic selection, PG3S+L‐, PERT or infectivity RT‐PCR assays are acceptable test methods.
If the viral preparation has been proven to be negative for RCV, animals injected with transduced cells may be housed at ABSL‐1.
18 IBC Testing Requirements for Viral Vectors
Viral preparations for in vitro experiments of short duration (used within 72 hours of viral particle generation): Every six months, a representative viral preparation must be tested to ensure RCVs are not being produced.
Every producer cell line or stably transduced cell line generated or placed in a laboratory repository must be tested for RCV prior to use.
If a viral preparation is being administered to animals directly, it must be tested for RCV. If cells are being transduced with the intent of administering the cells to animals, you must either test the viral preparation for RCV before transduction or test the cells for RCV following transduction.
Marker rescue, antibiotic selection, PG3S+L‐, PERT or infectivity RT‐PCR assays are acceptable test methods.
If the viral preparation has been proven to be negative for RCV, animals injected with transduced cells may be housed at ABSL‐1.
19 20 21 For more information on Adenovirus integration into host genome: http://www.ncbi.nlm.nih.gov/books/NBK8503/
22 People are frequently infected by adenovirus. If a researcher suffering from an adenovirus infection were exposed to a recombinant replication‐incompetent adenoviral vector, there is a potential for the wild type virus to complement the viral vector and allow for its replication and spread. This may have harmful effects if the rDNA carried by the vector is hazardous (toxin, oncogenic). Therefore, it is important to follow strict BSL‐2 precautions and suspend work when the researcher is feeling unwell.
23 IBC Testing Requirements for Viral Vectors
Every viral preparation must be tested for RCV by the E1a PCR assay prior to in vitro or in vivo use.
If the viral preparation has been proven to be negative for RCV, animals directly injected with virus or transduced cells may be housed at ABSL‐1 after 72 hours.
24 25 26 AAV can bind to enter many different animal cells including human, non‐human primate, canine, murine, and avian cell types. After entry into the cell, the virus will follow either a lytic or lysogenic path. In the absence of a helper virus, AAV will establish a lysogenic infection where the viral genome will persist within the host cell indefinitely but no viral progeny are produced. AAV will only replicate and produce viral progeny when in the lytic stage. This stage is dependent on the presence of helper viruses such as adenovirus or herpesvirus or helper functions provided in trans. If a host cell with a latent AAV infection is then infected by a helper virus, the AAV virus will switch to a lytic stage and replicate to produce viral progeny. These progeny are release upon helper virus‐induced cell lysis.
The dependence of AAV on helper virus in order to replicate plays a role in how the viral vector is packaged and produced. Original AAV packaging schemes required the presence of adenovirus for vector production. In this case, you can have helper virus contamination and possible rescue by co‐infection. AAV vectors produced in this way must be handled with the same cautions as when working with Adenovirus alone. More recent technology allows the Adnoviral genes needed for AAV replication (E1A, E1B, E2A, E4ORF6 and VA) to be provided on a plasmid in trans. This packaging scheme eliminates the possibility of helper virus contamination and thus the hazards associated with this Risk Group 2 agent.
27 IBC Testing Requirements for Viral Vectors
Adeno‐Associated Virus (with adenovirus helper) Every viral preparation must be tested for the presence of replication‐competent adenovirus prior to in vitro or in vivo use.
Viral preparations that have undergone heat‐inactivation may be tested for the presence of replication‐competent adenovirus by plaque assay or cytopathic effect. An E1a PCR assay may be used to test viral preparations purified with a heparin‐based sulfate column.
If the viral preparation has been proven to be negative for RCV, animals directly injected with virus or transduced cells may be housed at ABSL‐1 after 72 hours.
28 29 30 31 32 Why use a BSC if my bug is not transmitted via aerosol route?
Answer: Working in a BSC provides other advantages in addition to containment of aerosols. Utilization of a BSC confines the work space where biohazards are manipulated, reducing their accidental spread beyond the BSC. This confined space can often be easier to decontaminate than large open benches.
33 IBC Testing Requirements for Viral Vectors
Viral preparations for in vitro experiments of short duration (used within 72 hours of viral particle generation): Every six months, a representative viral preparation must be tested to ensure RCVs are not being produced.
Every producer cell line or stably transduced cell line generated or placed in a laboratory repository must be tested for RCV prior to use.
If a viral preparation is being administered to animals directly, it must be tested for RCV. If cells are being transduced with the intent of administering the cells to animals, you must either test the viral preparation for RCV before transduction or test the cells for RCV following transduction.
Serial transfer and p24 ELISA assay is the most common test method currently recommended by the IBC.
Other methods of testing not listed will be considered, provided the investigator presents sufficient rationale for the use of alternative methods, provides accurate details of the method (including what controls will be used) and cites published experimental validation of the method.
If the viral preparation has been proven to be negative for RCV, animals injected with transduced cells may be housed at ABSL‐1.
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