Use of Viral Vectors

Viral vectors are common tools used in molecular biology as a delivery system for genetic materials into cells that may normally be difficult to transfect. Viral vectors are constructed by replacing wild type viral genes with transgenes of interest.  This usually involves removal of viral replication genes  so that the vector is rendered safer than than the wild-type virus (i.e. viral vector is replication incompetent). While viral vectors are modified in such a way as to minimize the risks when handling them there still may be biosafety concerns associated with their use. These include:

• Replication Competence - While these vectors are designed to be replication deficient, it is impossible to completely control for the possibility of the generation of replication competent virus or reversion to wild type virus through recombination.
• Off Target Effects - For viral vectors that integrate into the host cell genome, off target effects such as insertional mutagenesis can be a safety concern.  Integration into genes that are important for cell division or programmed cell-death can potentially result in oncogene activation or tumor-suppressing gene inactivation.
• Transgene Hazards - The gene of interest can present a hazard itself. Transgenes that are known for oncogenic potential, encode apoptotic or toxin molecules present a higher biosafety risk and should be evaluated during the initial risk assessment prior to beginning work with these materials.

Viral vectors used in research involving recombinant techniques are regulated by the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (rsNAM). Project directors planning to conduct research with viral vectors and rsNAM must submit their research protocols to the Institutional Biosafety Committee (IBC) for review. For further information please visit the Office of Research Compliance website for biosafety.

The following are commonly used viral vectors along with required containment levels and other related safety information:

Adeno-associated Viruses (AAV)

Adeno-associated viruses are small, non-enveloped viruses with single strand linear DNA. They are members of the Parvoviridae family and there are 12 serotypes.  To replicate, AAV require helper viruses (wt adenovirus or herpesvirus).  In the absence of these helper viruses, AAV can stably integrate into the host cell genome.  AAV vectors are non-pathogenic and can infect dividing and non-dividing cells making them preferred viral vectors for many applications.

• Potential Health Hazards - While there are no known diseases associated with AAV human serotype, insertional mutagenesis is a theoretical possibility due to the ability of AAV to integrate into the host cell genome. 
• Laboratory Hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols
• Risk Group - Risk Group1
• Containment - BSL-1/ABSL-1 (Exceptions include presence of a helper virus then BSL-2 /ABSL-2; Packaging in human cell lines then BSL-2; Potentially hazardous transgenes such as oncogenes and toxins BSL-2/ABSL-2 )
• Disinfection/Decontamination - 0.5-1% sodium hypochlorite (10% Bleach), 2% glutaraldehyde, 0.25% sodium dodecyl sulfate, Autoclave (121°C, 1hr)

Adenoviruses

Adenoviruses are medium-sized, non-enveloped viruses with double-stranded linear DNA.  They are members of the Adenoviridae family and there are close to 100 different serotypes with over 40 being human pathogens.  Adenoviral vectors are non-integrating and are a popular choice in gene therapy due to their high transduction efficiencies, ability to infect many cell types, and high level of transgene expression.

• Potential Health Hazards - Exposure to adenovirus can cause a wide range of symptoms including bronchitis, sore throat, fever, diarrhea and pink eye.  Infections can be more severe in very young and immunocompromised individuals.
• Laboratory Hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols
• Risk Group - Risk Group 2
• Containment - BSL-2/ABSL-2. Adenovirus must be administered to animals under ABSL-2 containment. Animals must be housed under ABSL-2 containment for 72 hours after adenovirus administration, after which animals may be moved to ABSL-1 containment.
• Disinfection/Decontamination - 1% sodium hypochlorite (10% Bleach), 2% glutaraldehyde, 0.25% sodium dodecyl sulfate, Autoclave (121°C, 1hr)

Lentiviruses

Lentiviruses are enveloped retroviruses that are characterized by a long incubation period, immune evasion, and persistent infections in their natural hosts. They have single stranded and linear RNA that is reverse transcribed to produce DNA upon entry into the host cell.  This DNA transcript then integrates into the host's genome.  Lentiviruses have the ability to integrate into the genome of non-dividing cells, a feature that distinguishes lentiviruses from other retroviruses.  Adverse events can include leukemogenesis and oncogenesis through insertional mutagenesis. There are 5 recognized serotypes with HIV-derived vectors being the most commonly used lentiviruses in biomedical research.

• Potential Health Hazards - Infection with Lentivirus (HIV) can cause initial symptoms that are flu-like. Symptoms can be more severe in very young and immunocompromised individuals.  Infection is persistent and lifelong due to their ability to integrate into the host chromosome and the ability to evade host immunity.
• Laboratory Hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites) and ingestion. Aerosol transmission unknown. Major risks associated with HIV-1 based lentivirus vectors are the potential for generation of replication-competent lentivirus and potential for oncogenesis.
• Risk Group - Risk Group 3
• Containment - BSL-2/ABSL-2. Lentivirus must be administered to animals under ABSL-2 containment. Animals must be housed under ABSL-2 containment for 72 hours after adenovirus administration, after which animals may be moved to ABSL-1 containment.
• Disinfection/Decontamination - 0.5% - 1% sodium hypochlorite (10% Bleach), to a lesser extent 70% ethanol, UV light, pH higher or lower than the optimal level of 7.1, Autoclave (121°C, for at least 30 minutes)

Retrovirus / Moloney Murine Leukemia Viruses (MMLV)

Moloney murine leukemia virus (MMLV) is an enveloped single-stranded RNA gammaretrovirus that can cause cancer in mice.  MMLV can randomly integrate into the genome of a host cell. The host range of recombinant MMLV depends on the specificity of the viral envelope.  Ecotrophic vectors can only infect rodent cells whereas amphotrophic vectors can potentially infect a wide range of cell types.  

• Potential Health Hazards - Pseudotyped MMLV that infect human cells can result in insertional mutagenesis that can lead to development of malignancies.
• Laboratory Hazards - Mucus membrane exposure, contact with tissues and bodily fluids of animals, and parenteral inoculation (e.g. needle sticks and animal bites)
• Risk Group - Risk Group 1 (Ecotrophic), Risk Group 2 (Amphotrophic or pseudotyped)
• Containment - EcotropicMMLV  : BSL-1/ABSL-1.  Amphotropic or VSV-g pseudotyped vectors containing biological toxin or gene with oncogenic potential : BSL-2/ABSL-2
• Disinfection/Decontamination - 0.5% - 1% sodium hypochlorite (10% Bleach), 70% ethanol, 2% glutaraldehyde

Baculovirus

Baculovirus is a lytic, enveloped DNA virus that infects insects.  Baculovirus vectors are mainly used to produce high levels of recombinant proteins in insect cells as they have large cloning capacity and low cytotoxicity.  Wild type virus is non-pathogenic to humans but recombinant virus using specific promoters allows for gene expression in mammalian cell lines. 

• Potential Health Hazards - Certain transgenes (e.g. oncogenes, mammalian specific toxins) could potentially pose a health risk and must be taken into consideration when performing an initial risk assessment. 
• Laboratory Hazards - Direct contact, droplet exposure of the mucus membranes, parenteral inoculation
• Risk Group - Risk Group 1
• Containment - BSL-1/ABSL-1. Baculovirus pseudotyped with VSV-g can transduce human cell line s and should be handled at  BSL-2/ABSL-2.
• Disinfection/Decontamination -  10% Bleach, 70% ethanol, Autoclave (121°C, for at least 30 minutes)

Rabies Virus

Rabies virus is a neurotropic virus in the Rhabdoviridae family of viruses. It causes a common zoonotic infection from bats and other wild animals that results in encephalitis or paralysis and is often fatal.  It's neuronal tropism makes it a good candidate for studying neuronal trafficking  or to express genes efficiently in neurons.

• Potential Health Hazards - Infection with rabies virus can cause Flu-like symptoms, gastrointestinal, neurological and respiratory symptoms.  The virus can cause acute progressive encephalomyelitis. Once clinical signs of rabies infection manifest themselves, the disease cannot be curred or treated and is nearly always fatal.
• Laboratory Hazards - Mucus membrane exposure, parenteral inoculation (e.g. needlesticks and animal bites), ingestion and inhalation of aerosols
• Risk Group - Risk Group 2 (Recombinant vector). Rabies virus is Risk Group 3.
• Containment - BSL-2/ABSL-2 for most constructs but may require BSL-3/ABSL-3 depending on risk assessment
• Disinfection/Decontamination - 70% ethanol, phenol, formalin, ether, trypsin, some other detergents, UV light