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:
Keir M. Balla, Marlen C. Rice, James A. Gagnon, Nels C. Elde. Discovery of a prevalent picornavirus by visualizing zebrafish immune responses. BioRxiv 823989; doi: http://dx.doi.org/10.1101/823989
We will adhere to the Universal Principled (UP) Review guidelines proposed in:
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. (2019) Universal Principled Review: A Community-Driven Method to Improve Peer Review. Cell . 2019 Dec 12;179(7):1441-1445. doi: https://doi.org/10.1016/j.cell.2019.11.029
SUMMARY: The revolution in metagenomics has revealed the ubiquity of viruses in the environment. Viruses outnumber hosts, and only a small fraction cause disease. One way to identify potentially pathogenic viruses specifically associated with a host is to select for engagement of antiviral immune responses. Here, Balla KM, et al ., report on the development of a transgenic zebrafish line that produces green fluorescent protein (GFP) in response to the antiviral zebrafish type I interferon (IFN) protein, IFNφ. They observed spontaneous GFP expression in a minority of zebrafish only days after hatching. They employed RNA sequencing and 5’-RACE to identify the complete genome of a new picornavirus, ZfPV, in the GFP-expressing fish that was distantly related to known viruses. By conducting bioinformatic analyses on publicly available sequence data they identified ZfPV in seemingly asymptomatic fish in labs worldwide. They observed higher viral load in clonal CG2 zebrafish that have a single core MHC haplotype. They documented infection of the GI tract, as well as other tissues, but the natural history of infection remains to be determined. They confirmed authentic IFN responses in the GFP+ zebrafish by identifying increased expression of numerous interferon stimulated genes (ISGs).
OVERALL ASSESSMENT:
STRENGTHS: In general, the data presented adequately supports the authors’ conclusions. The manuscript is well written and the data is clearly presented. Attention is paid to controls and appropriate statistical tests are applied to demonstrate significance. The authors make good use of pre-existing datasets to add strength to their findings. Obviously, there is a strong element of discovery in the manuscript with the serendipitous discovery of a new zebrafish picornavirus. The discovery methods described in this manuscript may be employed in other model organisms.
WEAKNESSES: The primary weakness of the study was that the authors did not clearly establish causality. The conclusion could be strengthened by isolating the candidate picornavirus (using well-established methods for human picornaviruses) and transmission to a naïve zebrafish, and demonstration of replication and ISG production, which could be accomplished using RT-qPCR. To be clear, further characterization of the natural history of infection and host determinants is not required, but some demonstration of an infectious agent is necessary to support the authors’ conclusions. Other minor weaknesses are detailed below.
DETAILED U.P. ASSESSMENT:
OBJECTIVE CRITERIA (QUALITY)
Quality: Experiments (1–3 scale) SCORE = 1
Figure by figure, do experiments, as performed, have the proper controls?
Are specific analyses performed using methods that are consistent with answering the specific question?
Is there the appropriate technical expertise in the collection and analysis of data presented?
Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?
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.
Quality: Completeness (1–3 scale) SCORE = 2.5
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.
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.
Quality: Reproducibility (1–3 scale) SCORE = 2
Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?
Are methods for experimentation and analysis adequately outlined to permit reproducibility?
If a “discovery” dataset is used, has a “validation” cohort been assessed and/or has the issue of false discovery been addressed?
Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1
Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?
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?
Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.
MORE SUBJECTIVE CRITERIA (IMPACT)
Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1
A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A “1” would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.
How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.
Impact: Extensibility (1–4 or N/A scale) SCORE = N/A