Titrating Stocks of Recombinant Baculoviruses using the Plaque Assay Technique

We were going to devote this blog to a comprehensive account of how you might determine the infectious titre of your virus stock.  However, when our CEO got wind of this he started ruminating about how things were “back in the day” and how he had to collect seaweed from the seashore to extract agar for his overlays…!  No, we didn’t believe him either, but he is approaching his 7th decade so we cut him some slack!  But if you read on below maybe there is some truth in the rumour.  What this outburst of near senility did prompt was a consideration of the origins of the plaque assay method for determining the infectious titres of virus stocks.  So here goes.

The plaque assay of infectious virus is one of the oldest techniques in virology, dating from 1917 when Félix d’Herelle developed the technique to study bacteriophages, which infect bacteria.  It has to rank alongside the development of DNA sequencing or PCR for its impact on a field of study.  Possibly, it was the CRISPR of its day?  Essentially, the method depends on limiting the spread of virus from a single cell infection so that only surrounding cells become infected in subsequent rounds of replication.  This produces the characteristic hole in the cell sheet that is called the plaque.  The semi solid medium that limits virus spread is based on agar, which was originally discovered in Japan in 1658 after an innkeeper noticed that discarded seaweed soup solidified, according to legend1!

The method was modified in 1952 by Renato Dulbecco for eukaryotic cells and animal viruses; originally for polioviruses.  Dulbecco later was joint winner of the 1975 Nobel Prize in Physiology or Medicine for his work on tumour viruses.  In 1978 Martha Brown and Peter Faulkner published a method for the titration of baculoviruses in insect cells.  Despite the development of other techniques for estimating infectious virus titres, the plaque assay has remained a stalwart technique.  However, with the spread of baculovirus expression technology into fields other than virology, it is a method often regarded with trepidation and almost fear by many scientists.  We provide a detailed protocol for baculovirus plaque assays elsewhere (baculoCOMPLETE user manual) but here we consider some of the common problems encountered.

Perhaps the most important factor in producing good plaque assays is the nature and quality of the host insect cells.  For baculovirus expression vectors based on Autographa californica nucleopolyhedrovirus (AcMNPV) these assays are almost always done using Spodoptera frugiperda (Sf) cells.  These may comprise Sf21 cells propagated in TC100 containing 5-10% bovine serum or Sf9 cells grown in serum-free medium (eg ESF921 – link).  Although Sf9 cells are clonal (originally derived from Sf21 stocks) and are suggested to develop more uniform plaques, we regard Sf21 cells as superior for this method.  In our hands, Sf21 cells develop plaques faster than those from Sf9.  This may be a consequence of the superiority of Sf9 cells for recombinant protein production over Sf21 cells.  Sf9 cells seem to maintain their integrity for longer than uncloned Sf21 cells.  While an advantage for protein production, this may delay the formation of clear plaques, which depend on cell death and inability to take up a stain.

However, it may be impractical to keep both cell lines going in your laboratory, with Sf21 cells only needed for virus titrations.  Therefore, we have optimized the procedure for titration of viruses using Sf9 cells and suggest the following:

  • Whichever cell line is used, the cells harvested from either spinner (Sf21) or shake (Sf9) suspension cultures must be in mid log phase of growth and near 100% viable.
  • Cells may be used to seed culture dishes of your chosen dimension and should settle to form a sub confluent monolayer in less than an hour. Cells may also be used to seed dishes the day before use.
  • While individual culture dishes may be used, it is more convenient to use either 6, 12 or 24 well plates for plaque assays.
  • While the Sf9 cells are usually grown in serum-free medium, we recommend that the plaque assay titrations are performed in serum-containing medium, preferably TC100 with 10% FBS. The Sf9 cells seem to tolerate the abrupt transition to a new medium very well.
  • It is also better to dilute virus stocks prior to titration using TC100/10%FBS, as the high protein content prevents virus binding to plastic tubes and thus artificially lowering the infectious titre.
  • When titrating several virus stocks simultaneously, consider using 48-well plates for rapid virus dilutions. However, we shy away from using multi-channel pipettes as the tips seem to keep falling off!
  • Always use an overlay comprising low melting point agarose – insect cells will not tolerate regular agarose used for mammalian cells.
  • Avoid drying of the cell monolayer at any point in the plaque assay procedure as this results in cell death and holes in the monolayer. This is a particular problem when working in a class II hood with a strong downdraught.
  • Try to use microscopy grade neutral red for staining cell monolayers. We recommend Gurrs as superior to other brands.
  • If serum-free medium must be used for the virus titration, beware we have noted that the plaques that develop are prone to fade rapidly after staining the cell monolayers with neutral red.
  • If at first you don’t succeed, try, try and try again! It usually takes a few attempts to obtain really nice plaque assays, but when you get a good one it is almost a work of art! Our placement student struggled with plaque assays a little at first but now produces some really nice ones.

An example of a good quality plaque assay is shown below (Fig. 1).plaque 2

Figure 1.  Titration of wild type baculovirus in Sf21 cells.  a – c represent high to low dilutions of virus inoculums. Note how a plaque can sometimes appear to be merged with their neighbour.

We also offer a virus titration service using either the plaque assay or quantitative polymerase chain reaction method (link), which we will discuss in our next blog.

 

References

1Mary Jo Zimbro, David A. Power, Sharon M. Miller, George E. Wilson, Julie A. Johnson (eds.).Difco & BBL Manual PDF) (2nd ed.). Becton Dickinson and Company. p.6.