Write a comment

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: 

Morphology-dependent entry kinetics and spread of influenza A virus

Sarah Peterl, Carmen M. Lahr, Carl N. Schneider, Janis Meyer, Xenia Podlipensky, Vera Lechner, Maria Villiou, Larissa Eis, Steffen Klein, Charlotta Funaya, Elisabetta Ada Cavalcanti-Adam, Frederik Graw, Christine Selhuber-Unkel, Karl Rohr, Petr ChlandabioRxiv 2024.08.01.605992; doi: https://doi.org/10.1101/2024.08.01.605992

We will adhere to the Universal Principled (UP) Review guidelines proposed in: 

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029 

SUMMARY: Influenza A viruses (IAVs) display filamentous and spherical morphologies in nature, but functional differences between these morphologies remain largely understudied, in part because cell culture adaptations are typically accompanied by commitment to a spherical morphology. This report describes new research methods to study the biological significance of IAV spherical and filamentous morphologies in vitroand in vivo. Using reverse genetics, the authors created IAV WSN/33(H1N1) reporter viruses encoding mScarlet, with M1 genes that support either spherical or filamentous morphology, to enable direct comparisons of the biological effects of different virus morphologies on otherwise identical backgrounds. Using a variety of assays, they demonstrate that spherical virions enter cells and spread faster than filamentous virions. By contrast, filamentous virions, bearing more hemagglutinin copies per virion, were better able to evade neutralizing antibodies. These studies provide a starting point for further investigation using this approach.

OVERALL ASSESSMENT: This report describes functional differences between spherical and filamentous IAV morphologies in controlled settings, building upon previous studies. The central claims of the manuscript are generally well supported by the data. We identified some areas where quantitative analyses and data presentation can be strengthened.

STRENGTHS: The manuscript is generally well written. Discrete control of virion morphology through M1 mutagenesis was viewed as a strength, and these tools will undoubtedly be used by others to advance the field. For the most part, advanced microscopy methods yielded informative, high-quality data. Phenotypes were confirmed in multiple cell lines, demonstrating a certain extent of generalizability in the findings.

WEAKNESSES: The mScarlet reporter gene was lost from a significant proportion of viruses, as evidenced by plaque analysis, and mScarlet-deficient plaques were larger than mScarlet expressing plaques. The authors acknowledged this shortcoming but persisted in using these reporter viruses for the study, instead of further reporter gene optimization. As there were several assays that did not rely on the fluorescent reporter, reverting to reporter gene-deficient parental viruses could have been an alternative approach to power most of the study. Statements about numbers of viral particles in endosomes in Figure 3 would have been better supported by quantitative data rather than representative images. While it is interesting to observe filamentous viral particles in one endosome, this single observation does not provide sufficient insight to draw conclusions about the fate of filamentous virions in endosomes. The authors should clearly indicate the basis for estimates of number of HA proteins per virion, which are based on virion size rather than any direct measurements; we discussed the potential for flow virometry approaches for single virion analysis of HA levels. Some figures lacked statistical analysis, and overall, statistical methods could have been better described throughout.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments  

·     Figure by figure, do experiments, as performed, have the proper controls?

·     Overall, these experiments appear to be properly controlled.

·     Are specific analyses performed using methods that are consistent with answering the specific question?  

·     It is unclear on why specific cell lines were used for specific experiments (e.g. A549 cells in Figure 3 and MDCK cells elsewhere). Rationale for cell line choice should be provided.

·     The use of the fluorescent reporter virus, which was unstable and had a plaque formation defect relative to WT parental virus, was not properly justified. Further assay development and refinement could have mitigated these drawbacks for reporter viruses, or they could have been dispensed with entirely, as several assays did not require a reporter gene. 

·     Is there appropriate technical expertise in the collection and analysis of data presented?

·       Yes

·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?  

·   There were a number of instances where quantitative methods were not performed, which undermined the authors’ conclusions.

·   Flow virometry could provide a beneficial alternative approach to study HA content and particle morphology. Here are some relevant studies that demonstrate this approach:

https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpz1.368

https://journals.asm.org/doi/10.1128/jvi.01765-17

https://journals.asm.org/doi/10.1128/jvi.01717-24

·     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.  

·       Citation of one source to justify inference of HA content based on virion size in Figure 5. This approach could be better supported.

2.   Quality: Completeness  

·     Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. 

·       In the abstract, the authors state that they show “cellular junction integrity... do[es] not exert morphology-dependent inhibition of IAV cell-to-cell spread” – this would be in reference to Fig 4 B-C where they show that knocking out catenin doesn’t change the performance of the filamentous virions relative to the spherical. Is this really a measure of the importance of cellular junction integrity, or is it showing that even when cells are growing farther apart/at a lower density, the spherical virions are still better?

·     Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. 

·   N/A.

3. Quality: Reproducibility  

·     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

·       The observation of single endosome with filamentous particles in it (Fig. 3G) clearly does not adhere to these principles of quantitative analysis. The reader should be made aware that this was a single event and cannot be broadly interpreted as a generalizable phenotype without further validation.

·     Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

·       Yes

·     If a ‘‘discovery’ dataset is used, has a ‘‘validation’ cohort been assessed and/or has the issue of false discovery been addressed?  

·       N/A

4. Quality: Scholarship  

·     Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? 

·       In this system, a slightly higher concentration of stalk-binding antibody was required to inhibit filamentous viruses compared to spherical viruses, supporting the idea that the elevated levels of HA on filamentous viruses confers an advantage. However, the plaque size phenotype persists in the presence of anti-HA antibodies, whereby the spherical viruses make much larger plaques. Given these findings, a more nuanced discussion of the effects of anti-HA antibodies on the two viruses would be beneficial. Would polyclonal anti-HA antibodies in vivo have a different effect?

·     Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? 

·       We were unable to locate the citation for the previous study that demonstrated that filamentous virions contain more HA than spherical; this is important to justify the HA quantity estimation based on surface area in Figure 5A.

·     Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section.

·       Overall, the Results section would benefit from greater exposition and the strategic use of summaries to emphasize take-home messages.

·       We identified several aspects of scholarship for potential improvement, figure-by-figure:

·       Fig. 1:

·       1C: When referring to this figure in the Results text, the authors refer to five point mutations in M1; these are more properly described as amino acid substitutions, as there are certain to be many synonymous mutations throughout.  

·       1D/E: These representative images should have text indicating which virus is used. They are logically placed below cartoons, but since they are separate panels, the reader needs a little more guidance.

·       1D/E/G: Scale bars are present, but no values are shown, and reader must refer to figure legend. Clear scale bar values on the figure are better for the reader.

·       1F: This data could be more effectively presented, as the current plot of virion diameter vs. length obscures the magnitude of differences. A histogram or kernel density estimation plot to visualize the length distributions would be more appropriate. In the text they discuss the mean length for the reporter viruses, but it would be helpful to show variance and maybe a t-test.

Fig. 2. Statement in text “Uninfected cells showed numerous filopodia which have a wider and more variable diameter (69 ± 52 nm, n=10) than filamentous viruses” is not well supported by data. Quantitative methods should be clearly described.

Fig. 3. This data would benefit from more quantitative analysis, i.e. number of virions/endosome. This approach will reinforce details like the extremely rare observation of filamentous virions (in one endosome). Also, observation of filamentous virions in one endosome seems to be a rare event considering 50% of cells were infected. Detailed analysis of 3D segmentations in Figs 3I-L should be more clearly described in the text to help the reader understand significance. In Fig. 3B, even though there are few biological replicates, variance should be reported. An appropriate statistical test would support the conclusion that spherical viruses infected more cells than filamentous viruses.

Fig 4.

Fig. 4A. Including an actual picture of the cells here could be helpful in addition to the cartoon, to give the reader a true sense of the cell density. This is particularly important because the quantitative density analysis reveals a large spread of data.  

Fig 4F. It is unclear from figure and legend if these are mean radial profiles but from method and text discussing this figure leads to believe that these points are means, if so, there should also be a measure of variance included in the figure.

Fig. 4H is not currently mentioned in the Results text; the figure label should be “focus formation” not “plaque formation”.  

Fig 5. If authors are assuming that the filamentous virions have more HAs based on their size, then they should also maybe assume that there are more NAs. If there are more NAs, then this would have been relevant to mention in the section about the mucin experiments.  

MORE SUBJECTIVE CRITERIA (IMPACT): 

Impact: Novelty/Fundamental and Broad Interest  

How big of an advance would you consider the findings to be if fully supported but not extended? Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2)

·       We appreciated this approach to addressing knowledge gaps about the functional differences between spherical and filamentous virions. The authors position this study in context of the field, where in some cases, similar observations have been made in different systems (i.e. filamentous virions are better at escaping neutralization). This undermines the novelty of this study somewhat. Broadening this study to include an in vivo infection model (e.g. mice) would further extend these studies.

Competing interests

The authors declare that they have no competing interests.