A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

[…] Essential revisions:

1) All reviewers agree that there is insufficient evidence presented to distinguish between a 'stamping' model and others with a larger number of replicative events. The Discussion that begins in subsection “The mutation rates of influenza A virus” paragraph four is not backed up by data that the reviewer can access, and the conclusions of stamping vs. binary replication modes are sufficiently important that, unless more data are provided, this section should be deleted.

We recognize that our inference of linear vs. binary mode of replication relies on certain assumptions and concede that the evidence may be insufficient as presented. We also feel that the novelty of the manuscript lies elsewhere. Therefore, we have deleted the section of the Results and Discussion that deal with this question.

2) The virology presented here is complicated and well-designed. eLife has a wide audience that would benefit from more detailed explanation about the multiple-plasmid transfection to recover virus, the DeltaHA construct that then needs to be passaged in an HA-expressing line, etc. These details are in the Materials and methods, which is fine, but there are points that are relevant for the interpretation of the data. After transfection, for example, how long does it take for the first infectious cycle to complete? Is the 24-hour time point one such cycle?

We have added additional text in the Results, subsection “A fluorescence-based fluctuation test” to clarify how we used the 8 plasmid reverse genetics system to rescue recombinant viruses expressing GFP from transfected cells. We also clarified how these GFP viruses are only propagated on MDCK cells expressing HA in trans and how the use of non-HA expressing MDCK in the imaging plate allowed for infection, but not subsequent spread of GFP viruses.

With respect to the timing of the infectious cycle, we should be clear that none of the fluctuation tests were initiated with transfection. We used transfection of plasmids to rescue recombinant virus. This recombinant virus is a passage 0 (P0) stock. We then used this to infect cells and make a passage 1 (P1) stock, which was used for experiments. We usually harvest our P0 stocks ~ 48 hours after transfection. There is likely some degree of viral replication that takes place in this time period, but we have never quantified it.

We know from one step growth curves that the infectious cycle of PR8 is approximately 12-14 hours. Not surprisingly, the GFP virus grows slower (~17 hours), see for example Figure 2B. As indicated in the text, the timing of transfer from the replication plate to the imaging plate was determined empirically for each GFP virus (17-24 hours at 37˚C) to ensure an adequate Nf population size (both for adequate fluorescent imaging and to target a P₀ in the 0.1-0.7 range). Importantly, the number of infectious cycles on the replication plate is irrelevant to calculation of mutation rates using the null class model for fluctuation tests. The key parameters with respect to growth are the starting population (Ni) and the post-replication population size (Nf).

The imaging plate was fixed at 14 hours post infection. This time point was chosen as it was sufficient to allow for infection and expression of GFP from the genome of the transferred viruses used to infect the plate.

We have clarified these elements of experimental design in the Results section as above and in subsection “The mutation rates of influenza A virus”.

3) In Figure 2B, which mutant DeltaHA viruses are analyzed? Are the error bars because it is all mutant viruses, and is wild-type DeltaHA included? This experiment needs to be clarified.

This experiment had 4 replicates of the C-U mutant ΔHA-GFP virus and 4 replicates of the U-A mutant ΔHA-GFP viruses virus in the PR8 genetic backbone. The error bars are standard deviation for 8 replicates. We have clarified this in the figure legend “Data shown are the cumulative mean and standard deviations for 4 measurements at each time point for each of two mutant ΔHA-GFP viruses (C to U and U to A viruses). Each point is the therefore the mean ± standard deviation for 8 values.” We note that the fitness values derived from competition assay for 6 mutant ΔHA-GFP viruses are a better measurement of growth relative to the “wild type” ΔHA-GFP virus.

4) Subsection “A fluorescence-based fluctuation test” The phrase "high minimum free energy" will confuse some non-physical chemists. Perhaps 'suggests that the mutations are not located in highly stable RNA secondary structures' would be more helpful for making the point.

This is a good point. We have changed the wording as suggested, subsection “A fluorescence-based fluctuation test”.

5) Several improvements in clarity will improve the accessibility of the manuscript. For example, the 12 mutational classes are never defined. It would be good to do so in a few words (e.g., "all possible different single-nucleotide mutations"). The terminology of "missense" and "nonsense" mutations, while conventional, implies value statements. Greater clarity would be provided by "non-synonymous" and "mutation to stop codon". Note that the manuscript is somewhat inconsistent and uses both.

We have changed “12 mutational classes” to “all possible single nucleotide mutations” and “the twelve different mutation types” in the Abstract. We then define mutational classes more clearly at the first instance in the Introduction.

We have changed most of the “nonsense” references to “stop codons.” We have kept the term “nonsense mutational targets (NSMT)” as it has been used in the mutation rate literature and is more concise than “codons that are one mutation away from a stop codon.”

We verified that we were similarly consistent with non-synonymous and avoided the use “missense” throughout.