PONE-D-19-21391

The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation

PLOS ONE

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Academic Editor

PLOS ONE

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors characterize mutations derived from mouse adaptation of an H3N2 seasonal influenza virus strain.

In many places of the manuscript, the description is ambiguous and logically convoluted. There are also many blunders. The work is interesting, but experimental procedures and results need to be described more clearly and logically.

Major points:

1. A figure showing increase of viral titer in mice as the number of passage increases is recommended.

2. Was each mutation from each individual colony or all from a single colony? How many colonies were picked? In how many colonies was each mutation found? This information may be added to Table 1. If all from a single colony, different combinations of mutations in addition to single mutations should have been studied. Description of the process of identifying the mutations is recommended.

3. Growth curves of the subject viruses (mutants and MA_SW) in MDCK cells and A549 cells are recommended. This is to see if the mutations were simple growth adaptations or host adaptations. The authors mention viral growths in MDCK cells in many places of the manuscript without actually showing the data.

4. Since the input titers of the mutant viruses and MA_SW are different, the input titers of the mutants being 100-fold higher than MA_SW, it is difficult to determine the connection between each mutation and the MA characteristics. According to Fig 1 and 2, the mutants didn’t show MA characteristics at the titer where MA_SW was infected. Fig 3 and 4 cannot be interpreted properly for the same reason. To the reviewer, it appears that none of the mutations individually have notable MA characteristics. The authors didn’t make any effort to figure out which combination of the mutations recapitulates the MA_SW phenotype.

5. Since the virus of PA mutation alone was not rescuable, at least a combination of PA with other mutations could have been tried. Since the D34N of NP is frequently found in MA H3N2, combinations of the NP mutation with PA and PB2 mutations could have been tested. If all mutations were found in the same colony (there is no information about this in current manuscript!), please do the mutation combination experiments.

Minor points:

1. Is the amino acid position numbering a CDS numbering (not clear especially for HA)?

2. Line 146, title of Table 1 is wrong. Reorganizing the mutations by passage stage (i.e., passage in DBA/1J or Balb/c) is recommended.

3. Lines 152 – 157, please try to write more clearly.

4. Lines 168 – 169, description doesn’t agree with the data.

5. Lines 239 – 241, where is the reference?

6. Line 243 – 245, check ‘nine amino acid substitutions’ and ‘seroconversion’ if they are correct.

7. Lines 275 – 279, the authors appear to be mixed up about mouse and human.

Reviewer #2: This study analyzed genetic markers for virulence and adaptation of a mouse-adapted seasonal influenza H3N2 strains. Four reassortant viruses were generated through reverse genetics and were evaluated for their morbidity and mortality in a mouse model. The data suggested that HA mutations were the most pronounced markers for enhanced virulence. The claims are supported by the data, successfully determining the genetic markers for mouse adaptation and increased virulence of a human-infecting IAV. Overall, this study seems suitable for publication to the journal, but some minor revisions are suggested.

1. It would be great if passage numbers of appearance of mutations are presented. The order of mutations of each gene, especially HA, will provide a valuable insight into tracking mutations during the host adaptation.

2. Lines 275~279: The authors stated that NP mutant virus was safe and the mutation might be a strategy to attenuate the virus. However, Figure 3 and Figure 4 show that NP mutant virus has increased ability to replicate in several organs as compared to the parent virus. Also, given that the NP mutation was induced by adaptation process in mice, the mutation is likely to be associated with increased virulence. The statement should be rephrased.

3. The time-dependent growth kinetics of each mutant virus on a cell culture (such as MDCK cell) would provide a better interpretation on replication abilities of the viruses. Especially, it would be interesting to examine whether delayed replication of the PB2 mutant in vivo (Fig 3) can be reproduced in vitro cell culture.

4. Line: 152~154: What does this sentence mean "We found that the MA_SW titer was lower than that of other viruses (3.16×104 TCID50/mL)…? Does it mean that the virus did not replicate to high titer enough in cell culture? Please clarify the meaning of this sentence.

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Reviewer #1: No

Reviewer #2: No

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The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation

PONE-D-19-21391R1

Dear Dr. Han,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Man-Seong Park, Ph.D.

Academic Editor

PLOS ONE

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