Handling GMO and Pathogenic Virus Systems

All work involving GMO and pathogenic virus systems is of a higher risk to workers and the environment. Additional measures, complying with the standard PC2 systems, must be put in place when using these organisms.

General Procedures for cultures of, and/or cell lines harbouring, adenovirus or retrovirus.

The procedures necessary to control the risks, associated with handling these organisms, at an acceptable level can be time consuming. As with all Lab work you must allow sufficient time to work to appropriate safety standards.

• Hands must be washed before and after handling cell lines or viral preparations.
• Double gloving is recommended especially when using high concentration viral cultures.
• All work, involving opened vessels, must be done in a class 2 Biosafety Cabinet.
• Gloves, gown and eye protection must be worn at all times.
• Sharps are to avoided at all times. Plastic substitutes for hypodermic needles and glass pasteur pipettes must be used.
• The use of flip-top tubes should be avoided whenever practical.
• All culture vessels that do not have a liquid-tight seal must be bunded. If practical, only filter-top culture flasks should be used.
• Adenovirus, retrovirus-producer cells or transduced cells must be stored in designated incubators only. Retrovirally transduced cells may only leave designated incubators when verified free of infective virus.
• Centrifuging must be done in sealed buckets or rotors and the tubes extracted and opened in a class 2 Biosafety Cabinet.
• Aspirated liquid waste must be collected into a closed trap with disposal plastic liner. The trap must be pre-charged with sufficient Sodium Hypochlorite to give a Chlorine concentration of 1.25% when full of waste (this is a one in ten dilution of the Hypochlorite stocks). When the disposable liner is almost full a volume of 1.25 chlorine solution must be aspirated through the lines to disinfect them. The liner is then sealed mixed, allowed to stand for 15 mins and then disposed off.
• All waste must be collected in plastic biohazard autoclave bags, these must be sealed when full and placed in a yellow bin inside the laboratory.

• All work surfaces including Class 2 Biosafety Cabinets and equipment (e.g. pipettors, racks and microscopes) must be swabbed, after completion of work, with 1.25% chlorine solution and then 70% w/w ethanol. (Note: Chlorine is corrosive to metal it must be rinsed off with ethanol solution.).

Adenovirus Specific Requirements

The most commonly used strain: Type 5 Adenovirus, a double deletion mutant (AdEasy system) is highly infectious. It is generally considered a respiratory virus but is capably of infecting most cells. The genome remains in the cells as an episome (i.e. not integrated into the host genome) and hence is transient. Although this virus cannot replicate in the cells it infects, aerosols are still a potential hazard.

The following additional procedures apply to work with adenoviral systems:

• Centrifuging must be done in sealed buckets or rotors and the tubes extracted and opened in a class 2 Biosafety Cabinet. The buckets should be autoclaved after work is complete.
• All waste must be placed in a plastic autoclave bag inside a class 2 Biosafety Cabinet and then into a second autoclave bag and then directly into a yellow bin.

• All spills must be treated with 2.5% chlorine solution and the waste generated disposed off as above.

Handling Adeno-associated viruses

Wild-type adeno-associated virus (AAV) is a vector increasingly used for gene therapy work. Unlike retrovirus and adenovirus there is no known disease associated with AAV infection. AAV has a single stranded DNA genome that upon infection is converted into double stranded DNA and inserted randomly in the host genome. For replication AAV has an absolute requirement for a helper virus (e.g. adenovirus, herpes). The handling procedures for AAV are the same as those for Adenovirus.

Retrovirus Specific Requirements

Retroviral vectors have been developed for the efficient introduction of genes into animal cells. Classification of retroviruses is based on the species of host cells they can transduce. The recombinant retroviruses are deletion mutants in which the essential virus packaging genes have been removed and are provided either on separate plasmids or by the packaging cell line. Retroviruses function by infecting cells, converting their RNA genome into DNA and integrating it into the host genome i.e. "transduction". Consequently the genes persist in the transduced cells.

Ecotropic retroviruses:

Transduce rodent cells efficiently but cells of all other species to a very limited or undetectable level. Ecotropic viruses are commonly used in labs.

Amphotropic retroviruses:

Transduce cells from a wide range of species including humans and mice. As they are capable of transducing human cells they pose a higher risk to the worker.

Note: Due to risks of cross contamination, ecotropic and amphotropic cultures are not to be handled at the same time and must be kept in separate incubators. It is also advisable to use separate class 2 Biosafety Cabinets if possible.  If not, then a full end of work disinfection of the hood should occur between work with each type of culture.

The following additional procedures apply to work with adenoviral systems:

Ecotropic Retroviruses (non-infectious to Human cells)

Handled as for Adenovirus.

Amphotropic retroviruses (infectious to Human Cells)

• Centrifuging must be done in sealed buckets or rotors and the tubes extracted and opened in a class 2 Biosafety Cabinet. The buckets should be autoclaved after work is complete.
• Aspirated liquid waste must be collected into a closed trap with disposal plastic liner. The trap must be pre-charged with sufficient Sodium Hypochlorite to give a Chlorine concentration of 1.25% when full of waste (this is a one in ten dilution of the Hypochlorite stocks). When the disposable liner is almost full a volume of 1.25 chlorine solution must be aspirated through the lines to disinfect them. The liner is then sealed mixed, allowed to stand for overnight and then disposed off.
• All waste must be placed in a plastic autoclave bag inside a class 2 Biosafety Cabinet and then into a second autoclave bag and then directly into a yellow bin.

• All spills must be treated with 2.5% chlorine solution and the waste generated disposed off as above.

Fowlpox Virus Specific Requirements

Fowlpox virus (FPV), the prototypical member of the Avipoxvirus genus, infects chickens and turkeys.
Recombinant FPV vaccines expressing foreign antigens can be used to immunize animals.
As FVP can undergo abortive replication in mammalian cells, they can be used as a host range restricted mammalian expression vectors.
Proceed as for adenovirus with the following additional procedures:

• Substitute 3.0% Chlorine for 1.25% Chlorine in all decontamination procedures.

Note: Animal house managers will need to be informed about all microorganisms used in their facilities

Prepared by:Tony Richardson  14/06/2006
Reviewed by: Kate Dixon 06/07/2009