Gene Vector and Virus Core  

Care and Handling of Retroviruses

retovirus

It can be useful to think of retroviruses as small cells. They have a membrane, a small amount of cytoplasm and the capsid containing the genome is somewhat like a nucleus.  As such they are very unstable and should be treated gently like a cell would be.


DON'T:

  1. Don’t expose to environmental extremes (such as pH, temperature, organic solvents, protein denaturants, strong detergents, or cation chelators such as EDTA).  ALL liquids the virus is in (dilution buffers , cell culture media, etc.) should be pH= ~7.2.  Add 10 mM HEPES if in doubt to buffer the pH.
  2. In particular the VSV-G protein, which is on the surface of the virus and is required for infection, is extremely pH sensitive.  It loses about 10-fold in infectious activity for each 0.3 unit drop in pH below ~pH 7.0.  Although this effect is reversible to some degree the reversibility is time dependent.  Because of this pH sensitivity ALL liquids the virus is in (dilution buffers , cell culture media, etc.) should be pH= ~7.2.  Add 10 mM HEPES if in doubt to buffer the pH.
  3. Don’t expose to any condition that disrupts membranes (such as temperature, organic solvents, strong detergents).  This will result in near complete inactivation because an intact viral membrane is required for viral infection.
  4. Don’t introduce air into the virus by vortexing, blowing bubbles and similar operations which result in protein denaturation.  Denatured envelope proteins, which are required for infection, are inactive.
  5. Don’t dry.  Drying will also result in membrane disruption and near complete inactivation of the virus.
  6. Don’t freeze and thaw multiple times.  The titer may drop 2-3 fold (or more) with each freeze-thaw cycle.   Therefore it is best to avoid.
  7. Don’t expose to hydrophobic plastics (especially polystyrene) for prolonged periods.  Because retroviruses are surrounded by a mostly hydrophobic membrane they are very sticky and losses can occur if they are exposed to hydrophobic plastics while not frozen.  It is best to store thawed retroviruses in siliconized or low protein binding tubes and pipette it with similar pipette tips. 
  8. Don’t filter.  Filtering can reduce titer because viruses can stick to the filter.  If filtering is necessary it is essential that filters with 0.45 45 μm (or larger) pores are used.  Since the diameter of a retroviruses is about 0.15-0.2 μm, if a filter with 0.2 μm (or smaller) pores is used substantial loss of infectious titer can occur.

DO:

  1. Do aliquot and freeze at -80C for long term storage if the virus is not used within ~1 week.  Besides being intrinsically unstable it is possible for microorganisms to grow in residual cell culture media that is in the virus.  Freezing helps prevent this.
  2. Thaw on ice just before use.
  3. Use virus within ~ 6 months of storage at -80C unless the titer is much higher than needed.  Even when stored at -80C retroviruses will lose ~10x in titer every 6-12 months.

 

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