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The authors must respond to the minor corrections of the external reviewer and in addition I would ask them to respond to the following point: that although the authors have made good improvements for the suggested revision, regarding the point of clearly stating the objectives of the current manuscript they have still not clearly stated the novelty of their findings. I therefore make the following suggestion for a statement that can be inserted into the introduction; where the authors can give the following statement (or a modification of this) after they mention genetic drift- "Although most influenza evolution can be accounted for by genetic drift, there is also evidence of adaptive evolution of mutations which are under positive selection (insert your references of choice here). We have extended previous studies of basic similarity alignment scoring of similarity (insert your references of choice here) to now present a position specific analysis of glycosylation sites that are under selective pressure in influenza viruses."

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Academic Editor
PLOS ONE

Reviewers' comments:

Reviewer #1: The authors have made significant improvements to the manuscript, particularly the results. The manuscript can be accepted for publication once the following (minor) issues are addressed -

1. "Most current studies on the glycosylation of IVs mainly focus on a given strain or the specific subtype of HA or NA, with less consideration for the distributed regularity of the glycosites in whole HA and NA subtypes" - this is not entirely true. Other studies (e.g. Joshua L. Cherry et. al., PLoS Curr. 2009 August 18; Blackburne BP et. al., PLoS Pathog. 2008 May 2;4(5)) have analyzed the evolutionary dynamics of N-glycosylation sites of select subtypes or HA/NA as a whole. These studies should be duly cited.

2. Abstract: "Over the natural course of viral evolution, the distribution of glycosites in HA and NA should achieve a balance." - This is a vague statement that lacks any support. I suggest the sentence be modified (or removed) for clarity.

3. The authors conclude that select glycosites on HA and NA are universally conserved (esp. the 27NNST in H1 or the 30NGT in H8, 498NGT in H1 or 500NGS in H5, C-terminal of HA1). It would be appropriate if the authors can shed some light on the functional role of these ultra-conserved sites.

4. "The remainder of the current 16 NA subtypes are concentrated into two evolutionary groups" - I believe what is meant here is HA and not NA (minor typo maybe).

5. Since H3 is considered to be the most dynamic HA subtype in terms of glycosite diversity, it would be fitting if the authors can add a paragraph describing the glycosite variability in H3.

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