Biosafety

Biosafety of Retroviral Vectors

Adapted from: http://www.boneandcancer.org/UCMOLab%20Biosafety%20Manual_3-24-05.pdf

Biosafety of retroviral vectors: Retroviral vectors are becoming standard tools in cell biology as well as potential therapeutic agents for human disease. Many investigators have come to believe that retroviral vectors are safe, but current biosafety guidelines and distributors of vectors both recommend using the vectors under biosafety level-2 containment (BSL-2). The safety of retroviral vectors for both introduction into humans and for use in basic research continues to be an important issue. Production of retroviral vectors has a common strategy, although details may vary. Retroviruses package RNA molecules into virus particles. Normally, the double stranded RNA retrovirus genome is packaged into virions, but retrovirus packaging cell lines (also known as helper cells) are constructed in order to package other RNA molecules. These RNA molecules have limited retroviral sequences and commonly express a messenger RNA of interest (the "vector sequence") as well as a selectable marker such as a drug-resistance gene. A typical packaging cell line is stably transfected with two partial (split) retroviral genomes. One construct contains the gag/pol region that encodes proteins required for virus particle assembly and reverse transcription (copying the double stranded RNA insert into DNA), and the second construct contains the env gene that encodes the proteins needed for virus binding to, and entry into, target cells. The viral RNA encoding these functions is not packaged into virus particles because the RNA sequences needed for binding to gag proteins (the packaging signal, or Ψ) have been deleted. The vector sequence containing the packaging signal is transfected into the packaging cells, and inclusion of the packaging signal in the construct insures that the vector sequence is packaged into virus particles.

Virus particles are harvested from packaging cell lines transfected with a vector sequence, and these particles are used to "transduce" the vector sequence (as well as the retrovirus RNA) into target cells bearing the appropriate receptors for the retroviral or other viral envelope expressed on the virus particles. "Transduction" is in essence a one time infection since the viral particles are infectious, but their genetic information is insufficient to generate new infectious virus unless some rare rescue event takes place. Because none of the retrovirus genomes expressed in packaging cell lines is intact, no replication competent viruses are produced unless a rare and specific recombination event generates an intact retroviral genome. The virus particles are "infectious" for only one replication cycle. They can bind and enter target cells expressing appropriate receptors, although very low levels of virus entry may occur in the absence of specific receptor binding. The vector sequence is reverse transcribed into DNA, and the two retroviral LTR and the viral integrase mediate integration of the vector sequence into the target cell DNA. The integrated vector DNA becomes a permanent part of the target cell genome, and it is thus possible that rescue of RCR by recombination with endogenous retroviral elements can occur many years after the initial transduction of target cells. Generation of replication competent retroviruses (RCR) in target cells or tissues is the primary risk associated with the use of retroviral vectors. Assessment of this risk is the primary task in determining the safety of retroviral vectors. The target cell range of the vector is also a safety issue. Incorporation of a virus envelope that can infect cells from multiple species increases the risk of both RCR generation and the potential danger of any resulting virus, which could spread from one species to another. Another consideration in risk assessment is the nature of the vector coding sequence. Marker genes such as green fluorescent protein (GFP) pose no special risk (unless one is concerned with turning the proverbial green thumb into a literal green thumb). However, vectors that include genes involved in oncogenesis, growth regulation, innate or adaptive immunity, or infectious diseases obviously carry a greater risk. A strong oncogene (e.g., ras) in a vector that is later rescued into an RCR by recombination events would recreate a human version of mouse leukemia viruses. The widespread availability of retrovirus vectors for laboratory use and the generation of "safer" vectors appear to have resulted in a sense of false security, particularly amongst first time users of vectors. Thus, Biosafety level 2 containment and careful monitoring of long-term or animal experiments for the emergence of vector-derived RCR is essential to ensure safety.

In the Gene Delivery Core, we will only work the widely used retroviral vectors or vectors that were derived from them at the Core. To minimize the possible generation of RCRs, we will use transfection of the Phoenix ampho packaging cell line. Briefly, Phoenix ampho cells will be transfected with the retroviral shuttle vectors containing the transgenes. As a result, amphotropic retroviral particles will be produced so that they can be used to infect both rodent and human cells. No retroviral vectors will be used for direct injections into animals. Many mishaps can be avoided by being familiar with the protocols before undertaking experiments.

In addition to the guidelines for handling Biosafety Level 2 biohazardous materials outlined in Section I, the following precautions apply:

a. Always wear gloves whenever handling cell and tissue culture containers and the virus itself. Double gloves may be needed in the case of injecting viruses into animals.
b. Use the biosafety cell/tissue culture hood whenever handling cell and tissue cultures or pipetting the virus. The virus itself will only be handled in a biosafety level 2 cabinet.
c. Disinfect contaminated tips and waste with bleach (e.g., waste such as used pipettes, pipette tips, microfuge tubes, cell culture flasks will be first soaked in a bleach containing-beaker for 30 min. and then disposed to biohazard waste container. The work surface area will be disinfected by wiping with bleach
spray, followed by cleaning with 70% ethanol).
d. Clean the pipet aid and pipetman used for the experiment;
e. Properly dispose the disinfected waste into the biohazard bags.

Additionally, the following frequently asked questions may help you better understand retroviral vectors.

What disease could retrovirus cause? MoMLV is a murine retrovirus, and no known human diseases have been associated with this virus. However, the risks involving in the use of the retroviral vectors largely depend on the transgenes to be expressed. If potential oncogenic genes are used, special precautions have to be given how to handle the viral vectors.

How is it spread naturally? By direct contact or blood-borne.

When is it present in the animal facility? No retroviral vectors will be directly injected into animals. Rather, only the retroviral vector-transduced cells will be injected into animals.

How could I be exposed? By direct contact with the skin, or internal exposure through cuts or needle sticks.

What do I do to protect myself? Always wear gloves; Handle sharps carefully; Masks or biosafety laminar air flow hoods should be utilized when the virus is being handled.

What is an exposure? Direct contact with the skin, or any internal exposure through cuts or needle sticks.

What do I do if I am exposed? The affected areas will be thoroughly washed with soap and water. Notify Dr. He or Dr. Haydon.

Is it treatable? No virus-specific therapy is available although serious illness can be managed by treating symptoms and complications of the infection.

Should I be tested for it before or after an exposure? No standard tests and/or surveillance methods are available to determine possible retroviral vector infection.

Is there anyone who should not go into a room with this agent? Yes, Severely immunocompromised individuals. For example, individuals with advanced AIDS, transplant patients on large doses of steroids or other anti-immune medications.

Adenovirus Fact Sheet

This fact sheet applies to any work with adenovirus, adenoviral vectors, and Adeno-Associated Virus (AAV). Work with these agents requires implementation of BSL-2 practices and procedures PLUS precautions detailed below (BSL-2 + Adeno). Prior to commencing any work with these agents, a protocol must be approved by the Institutional Biosafety Committee (IBC) .
For a description of BSL-2 procedures, refer to CDC's Biosafety in Microbiological and Biomedical Laboratories.

Background

Adenovirus is a pathogen of respiratory and gastrointestinal mucous and eye membranes.
Adenovirus (replication deficient and replication competent) can cause corneal and conjunctival damage. Eye protection (goggles) must be worn when working with this agent/vector.
Adenovirus (unlike HIV or herpes) is quite stable. After having been extracted with ether and/or chloroform, it can still be infective.
The replication-defective virus may be complemented in vivo thereby causing the vector to become replication competent.

Mode of Transmission
Adenovirus may be transmitted by:

Droplet
Aerosol
Injection

Symptoms of Exposure
Any of these symptoms may occur following adenovirus exposure:

Acute respiratory illness (cold like symptoms)
Pneumonia
Conjunctival infection (red eye)
Corneal inflammation leading up to scarification

Laboratory Practices

NO work with adenovirus is permitted on the open bench. A Biosafety Cabinet must be used for all manipulations including (but not limited to):
- Pipetting
- Harvesting infected cells for RNA
- Loading and opening containers
  - Exemptions will be considered on a case by case basis by the Institutional Biosafety Committee. Within the Biological Safety Cabinet (BSC):
    - Plastic pipet tips and serological pipettes are to be placed in a cardboard box (ex. Fisher catalog number: 14-375-268) prior to disposal.
    - This box is placed into an autoclave bag with any other biohazardous waste in the BSC. The bag is to be closed and then sprayed with a disinfectant approved for efficacy against adenovirus (5% phenol or 10% household bleach).
    - The disinfected bag is then placed into a clean autoclave bag, and subsequently autoclaved for 1 hour at 121°C or 250°F (15 lbs per square inch of steam pressure).
All vacuum lines must be fitted with a hydrophobic and HEPA filter. (ex. Fisher catalog Numbers: 09-744-75, SLFG 050 10, 09-730-211).
Biohazard waste containers should be hard-sided with a foot operated lid, and easily decontaminated with liquid disinfectant. The autoclave bag should be folded over the rim so that only the inside of the bag is visible.
All centrifugation must be done in closed containers using sealed rotors.
Signs and labels must be placed to indicate each area where Adenovirus is used or stored (biological safety cabinets, incubators, refrigerators, laboratory entrance doors, etc.).
Laboratory must have negative air pressure relative to the hallway (confirmed during lab audit).
Laboratory gowns should be water resistant and have cotton knit cuffs with back closure and wrap around ties (ex. Fisher catalog numbers: 19 999 344A, and 17-988-496A).
The most effective germicides (with a minimum 15 min. contact time) are:
- Phenol (5%)
- Sodium hypochlorite (household bleach diluted to 200 ppm or 10%)
Adeno-associated viral vectors must be tested for presence of replication competent adenovirus after heat inactivation.
Respiratory protection is necessary for adenoviral work. If experiment parameters make it impossible to work with adenovirus within a laminar flow biosafety cabinet, a respiratory protection program must be in place and N95 fit-tested respirators must be worn. Contact Institutional Biosafety Committee.
Gloves must be worn at all times when working with adenovirus. Remove gloves using the inside-out technique. Dispose of gloves into biohazard waste container to be autoclaved. Wash hands immediately after removing gloves and before leaving work area. Never wear gloves outside of the laboratory, or touch doorknobs, telephones, personal belongings, etc. with gloved hands.
All cultures, stocks, or materials used to manipulate or otherwise exposed to adenovirus must be autoclaved prior to disposal (except sharps). Autoclave conditions to be met: 1 hour at 121°C or 250°F (15 lbs per square inch of steam pressure). The outside of sharps buckets must be decontaminated before disposal by incineration.

Animal Use

Concurrent approvals are needed from UK Institutional Biosafety Committee and UK Institutional Animal Care and Use Committee prior to commencing animal work with adenovirus.
Animals must be handled in a BSL-2 area designated and approved for adenoviral work.
Infected animals may excrete (shed) adenovirus (especially the first 72 hours after infection). Precautions must be taken not to create aerosols when emptying animal waste material, washing cages, or cleaning the room.
Special training must be given to all animal husbandry personnel on adenovirus. This training must address the hazards associated with the work, required practices and procedures and proper handling of bedding, cage washing, and all other husbandry materials associated with the experiment.
All necropsy must be performed in a necropsy room using Animal BSL-2 +Adenovirus precautionary practices and procedures.
Only lab personnel or animal husbandry workers trained to handle animals infected with adenovirus should be responsible for animal husbandry practices during the first 72 hours following infection of the animal.

Employee Exposure
Eye exposure from splash or aerosol:
Rinse a minimum of 15 minutes in eye wash or flush with water. Notify the Principal Investigator or Laboratory Supervisor, who will immediately contact Occupational Health Physician located in Student Health Services and direct the exposed employee to appropriate medical treatment at University Health Services, and to report the incident.

Needlestick, sharps exposure or non-intact skin exposure:
Contaminated skin should be scrubbed for approximately 20 minutes using a 10% povidone iodine solution (such as Betadine®) and copious amounts of water. Notify The Principal Investigator or Laboratory Manager, who will immediately contact Occupational Health Physician located in Student Health Services and direct the exposed employee to appropriate medical treatment at University Health Services, and to report the incident.

Adenovirus Material Safety Data Sheet

Name: Adenovirus type C strain 5 and recombinant vectors based on Adenovirus 5
Characteristics: Adenoviridae; non-enveloped, icosahedral virions, 75-100 nm diameter, double stranded, linear DNA genome. Virus is lytic.
Biosafety Level: NIH BSL 2

Health Hazard
Pathogenicity: Wild type Adenovirus infection varies in clinical manifestation and severity; symptoms include fever, rhinitis, pharyngitis, cough and conjunctivitis. The risk from infection by defective recombinant adenoviral vectors depends both on the dose of virus and on the nature of the transgene. Adenovirus does not integrate in to the host cell genome but can produce a strong immune response.
Host Range: Humans are the natural reservoir for wild type Adenovirus 5. Recombinant Adenovirus vectors infecta variety of mammalian cell types.
Mode of Transmission: Wild type virus is spread directly by oral contact and droplet spread; indirectly by handkerchiefs, eating utensils and other articles freshly soiled with respiratory discharge of an infected person. In the laboratory, care must be taken to avoid spread of infectious material by aerosol, direct contact or accidental injection.
Incubation Period: From 1-10 days

Viability
Drug susceptibility: No specific anti-viral available.
Susceptibility to Disinfectants: Susceptible to 1% sodium hypochlorite, 2% glutaraldehyde. Recommend fresh solution of 10% bleach for 30 minutes.
Physical Inactivation: Sensitive to heat; 1 hour at 56oC is used to inactivate virus.
Survival Outside of Host: Adenovirus has been reported to survive 3-8 weeks on environmental surfaces at room temperature.

Medical
Surveillance: Pre-employment serum samples banked. Monitor for symptoms; confirm infection by serological analysis or viral culture.
First Aid/Treatment: For splashes to the eye of material containing virus, rinse eye at eyewash for 15 minutes then report to hospital emergency room for evaluation. A serum sample should be taken as soon as possible (at UCSD this is done by Occupational Health). In the case of accidental injection of material containing virus, wash area well with soap and water then contact office of Occupational Health for advice, evaluation and serum sample. At UCSD,
notify supervisor and EH&S as soon as possible after exposure. Supportive therapy is indicated for symptoms of suspected infections.
Immunization: None available.
Prophylaxis: None available.

laboratory Hazards
Laboratory- acquired infections: Rare cases reported in laboratories working with clinical specimens.
Sources/Specimens: Respiratory secretions. Theoretical risk from exposure to laboratory cultures of wild type virus or recombinant virus.
Primary Hazards: Ingestion, droplet exposure of the mucous membranes, direct injection.
Special Hazards: Contact with feces or urine from infected animals for 72 hours post infection.

Recommended Precautions
Containment Requirements: Biosafety level 2 plus UCSD Adeno special practices and BSL 2 containment facilities for all activities involving the virus, recombinant virus vectors, and potentially infectious body fluids or Biosafety Manual for Molecular Oncology Laboratory 7 tissues.
Protective Clothing: Laboratory coat, gloves, goggles.

Handling Information
Spills: Allow aerosols to settle for 15 minutes; wear protective clothing and gently cover the spill with adsorbent paper towel and apply freshly prepared 10% sodium hypochlorite starting at the perimeter and working towards the center; allow at least 30 minutes contact time before clean up.
Disposal: Decontaminate all wastes before disposal; steam sterilization, incineration, chemical disinfection. At UCSD contaminated material may be sealed in labeled, doubled, red biohazard bags and transported in covered, leak proof containers to BFI disposal bins for eventual incineration.
Storage: In sealed containers that are appropriately labeled and in approved locations for BSL2 materials at -70C.
Transport: Material must be sealed in primary and secondary containers, appropriately labeled.

Transgenes and Other Foreign Genetic Elements
Considerations: What is the replication status of your vector? In general, recombinant Ad 5 vectors produced in the Vector Development laboratory are replication incompetent. What is the nature of the transgene/s - are any potentially hazardous transgenes expressed, i.e.. toxins, oncogenes? Have any foreign elements been introduced which alter the specificity, host range, stability, or titer of the resulting vector? It is imperative that those handling recombinant vectors consider both the nature of the virus used as a vector and the effects of any transgene, introduced genetic elements, or other modification.