Guidelines for working with retroviruses
Retroviruses
A virus containing an RNA genome is called a retrovirus. The RNA genome contains a gene which encodes the reverse transcriptase enzyme. After the retrovirus has infected a cell, the enzyme is used to perform reverse transcription of the RNA into DNA. This DNA is then spliced together with the cell's DNA, thereby forcing the cell to create a new virus. A special type of retrovirus exists which never leaves the cell it has infected, but instead becomes permanently enveloped in the cell’s genome. Such retroviruses are called endogenous retroviruses.
Retroviruses can be used to transport both known and unknown genes into different types of cells (infection). But these viruses are then mutated in the genes in question, which makes them able to infect only once (a life cycle). This will prevent the development and spread of replicating retroviruses. They usually have defective packaging signals or defective packaging signals and 3`UTR so that a triple recombinant event between the vector, or endogenous sequences and helper virus are needed in order to form replicating “independent” virus particles. Such an event has not yet been proven but we should nevertheless take certain precautions when working with retroviruses.
Types of retrovirus
Ecotropic retroviruses
Infect cells that express the ecotropic receptor, a murine large transmembranal protein which recognises the retroviral envelope protein of the Moloney murine leukaemia virus (MMLV). The majorities of murine and rat cells express this receptor and are therefore capable of becoming infected by viruses with this type of envelope protein.
By definition, ecotropic retroviruses are incapable of infecting humans. However, all work involving ecotropic viruses, hazardous genes or unknown viruses must be handled as amphotropic retroviruses.
Amphotropic retroviruses
Infects mammalian cell types, including human cells and requires that the retrovirale virus contains amphotropic envelope proteins. Lentiviral vectors are based on the fact that HIV virus is an example of an amphotropic retrovirus that is frequently used as a viral vector in research. Since, by definition, amphotropic virus is capable of infecting human cells and thereafter being integrated in the genome, one has to be very careful when working with such viral vectors. Cell culture work involving use of amphotropic retrovirus is therefore potentially dangerous, and this is especially related to use of vectors that contain proto-oncogenes and pathologically-related genes. In a worst-case situation can a user of such viral vectors develop cancer or some other illness following exposure to these recombinant retroviruses. Simple, but strict precautionary measures can prevent you, and those around you, from being exposed to this potential danger. In addition one should, on a regular basis, check the ability of the retrovirus being used to replicate to ensure that it cannot be spread.
When using lentivirus it is an advantage that a user endeavours to utilize a 3rd generation lentiviral vector. A 3rd generation lentiviral vector is safer for the user since essential genes that are necessary to make a non-replication proficient lentivirus are packed in three separate plasmids, which will in turn lead to a lower risk that replication competent retrovirus is produced.
Demands to room and equipment
For work with retrovirus a cell lab with biosafety level 2 or higher has to be used (BSL2, BSL 2+ or BSL3). If available then a lab with underpressure is preferred for performing work with retrovirus. A LAF-bench equipped with a UV-light, and a UV-light mounted in the ceiling that can be switched on after work with retrovirus in a cell lab is advantageous.
Laf-benches
All work with retrovirus shall be performed in a laminar flow hood equipped with an extraction filter/ HEPA-filter. A separate incubator shall be used for work with virus. The work bench shall be decontaminated with 70% ethanol before and after use, and at least once a day, and immediately after a spill. After a spill the bench plates are lifted up, and all surfaces are decontaminated with ethanol. After work the bench is to be washed with 70% ethanol. 70% ethanol is sprayed into the air in the cabinet in order to destroy eventual virus particles in circulation. Finally the UV-light in the LAF-bench is switched on.
Infected material
All infected material, medium and cells, are collected in separate closed flasks/containers and decontaminated with 25% chloramine, (a suitable alternative is 1% Virkon), for up to 24 hr in a work bench. The containers are sprayed with 70% ethanol before they are removed from the bench and placed in special waste-cartons («the yellow boxes») that are sent for destruction. When a vacuum-pump has been used with infected cells and medium make sure that there is no medium left in the tube and always hold the tube-end in the work bench. Wash out the tube with 70% ethanol after use, and then keep it immersed in 70% ethanol. Waste is to be autoclaved, and is then treated as ordinary cell lab waste.
Equipment
efore starting work, you must ensure that the following objects are in the bench or within arm’s length: a spray bottle containing 70% ethanol, a bottle of 25% chloramine, waste bags, necessary pipettes, a bucket containing 25% chloramine and other necessary equipment (media, PBS, cell bottles etc.).
Only disposable (plastic) equipment is to be used when working with infected material. All pipettes, tips and other equipment that has been in contact with retrovirus is to be treated with 25% chloramine before being decontaminated in 25% chloramine for 24 hr. If waste has to be removed from the LAF-bench immediately after rinsing in the chloramine-solution, it is to be placed in a separate autoclaving bag that is sealed in the work bench. The bag is sprayed with 70% ethanol before being removed from the bench. It is then placed in a «yellow box» that is sent for destruction.
Contaminated needles are disinfected in 25% chloramine, and then deposited in a separate plastic security-container. If one has to use reusable equipment then this must be sprayed with 25% chloramine (or 1% Virkon), and after 2 minutes this can be dried off. Thereafter the equipment is to be autoclaved.
Physical protection
When working with retrovirus one must use a facemask, hood, gloves and your own laboratory coat that can be fastened at the back. Gloves are to be deposited in a «yellow box» that is sent for destruction and hands are to be washed thoroughly before leaving the room. The doors to the cell laboratories must always be closed. Before leaving the cell laboratory all gloves, facemasks and lab coats are to be removed. If you are to work with amphotrope retrovirus then you shall in addition wear a double pair of gloves. Nitrile-gloves are preferable when performing this type of work. Having finished your work the outer pair of gloves should be deposited in an autoclave bag in the work bench as described above. The bag is placed in a «yellow box» which is sent for destruction.
Inspection of replicating retroviruses
Better safe than sorry!
The retroviruses should be regularly inspected to prevent the formation and spread of reproducible retroviruses. Media from secondary cultures (i.e. transfected cells) are used to transfect 3T3 cells. These cells are then selected for the relevant selection marker (usually G418). Media from the transfected 3T3 cells are then screened for reverse transcriptase (using a reverse transcriptase assay). Do not hesitate to ask if anything is unclear or there is something you do not understand.
If work with retrovirus has been performed in a BSL3 lab, it is not desirable that work in this laboratory is later carried out using replication-competent virus and gene modified vectors that are compatible with replication competent virus. This is to prevent generation of replication-capable gene modified virus.
See Cell- and tissue culture regarding general work involving cells and BSL2/3 work.