Cell . 2019 Dec 12;179(7):1441-1445. doi:
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?
- The experiments are properly controlled throughout.
- Are specific analyses performed using methods that are consistent with
answering the specific question?
- The bioinformatic analysis methods are largely consistent with
standards in the field. Generating phylogenies with different
methods (Fig. 2 – supplement 2) was considered a strength of the
study.
- One minor point is that the sequencing depth of non-zebrafish reads
is relatively low. There were 175 million starting reads, but only
8% of these were non-zebrafish, with 14 million remaining. Most of
these remaining reads mapped to bacteria or were unknown so in terms
of truly discovering novel viruses they really only had the power to
detect abundant viruses in these experiments. This clearly was
sufficient to identify ZfPv, but it would have been nice to get at
the whole virome with higher sequencing depth. In this regard, it
would be helpful if the authors could address potential limitations
of their analysis in more detail.
- Is there the appropriate technical expertise in the collection and
analysis of data presented?
- The experimentation and collection of data was generally of high
quality throughout.
- In the Methods section, it would be helpful if the authors could
clarify their reasoning behind pooling 30 GFP positive fish to
harvest RNA for sequencing.
- Do analyses use the best-possible (most unambiguous) available methods
quantified via appropriate statistical comparisons?
- For the most part, appropriate statistical comparisons are made
throughout.
- In Figure 4, RT-qPCR data was displayed as relative ZfPV levels on a
log scale y-axis by normalizing to lowest non-zero amount of ZfPV
RNA. This is common practice in the field. However, the unpaired
t-test assumes equal variances which may not be the case. The
analysis could be strengthened by determining whether the data is
normally distributed (e.g. Shapiro-Wilk test). If the data is not
normally distributed, then a non-parametric test (e.g. Wilcoxon
test) could be performed.
- 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.
- The experimental foundations of the study are generally solid.
- FIG 1: As a minor point,
there was discussion in the group about the merits of the dual
promoter construct driving eGFP from the cmlc2 (cardiac) promoter or
the IFN-inducible ISG15 promoter. There was concern that using the
same fluorescent protein for both promoters made it impossible to
determine IFN-inducible gene expression in cardiac tissue. However,
the counterargument was that the levels of eGFP in the cardiac
tissue could provide a useful reference for IFN-inducible eGFP
expression throughout the fish, acting as a normalization control
for fluorescence images. Some additional rationale describing the
merits of the expression construct would clarify this point.
- FIG 5: There was also discussion about the strength of the evidence
for a virus-specific response to ZfPV infection, by comparison toM. marinum infection. Does the fact that 12 or 13 genes are
induced in response to ZfPV infection, but not M. marinuminfection, truly represent a specific signature? The authors may
choose to soften this description until more data becomes available
in support of a ZfPV-specific host response signature.
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.
- Overall, the data generally supports the conclusions as stated in
the title and abstract, but there was significant discussion in this
room full of microbiologists-in-training of the merits of in
silico discovery of a new virus vs. discovery methods that adhere
to Koch’s postulates or Falkow’s molecular Koch’s postulates. There
was consensus that isolation of the virus and experimental
inoculation of naïve zebrafish and characterization of responses
would add great value to the study. RT-qPCR could be performed to
detect viral transcripts and ISGs in the same infected fish. Such
experiments would provide greater confidence that ZfPV is truly the
etiologic agent. Subsequent experiments to fully characterize immune
responses in the fish and host genetic determinants are beyond the
scope of this first report, but establishing causality is essential.
- For Figure 3, there was some discussion about the extent to which
the findings of ZfPV RNA in different tissues could be influenced by
dissection technique.
- In the absence of these experiments, the authors should acknowledge
these limitations and mention future research directions in the
Discussion.
- 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.
- See above re: virus isolation and infection of naïve animals.
- Furthermore, there was some concern about independent validation of
the RNA-seq experiments. Can ZfPV RNA be recovered from GFP-negative
animals? The RNA-seq data could be validated on samples of RNA from
individual fish using RT-qPCR and virus gene-specific primers. What
does it mean if ZfPV can be recovered from GFP-negative animals?
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.?
- There was considerable discussion about whether the sample sizes in
Figure 4 were sufficient to support the conclusions, but the group
was not able to come to consensus on this point.
- Is there sufficient raw data presented to assess rigor of the
analysis?
- Yes, although the uploading of raw data is still in progress at this
time. The dataset could be strengthened by making the video publicly
available.
Are methods for experimentation and analysis adequately outlined to
permit reproducibility?
- The bioinformatics methods are clearly described, but the scripts
should be made available on GitHub.
- There should be a description of how the genome viewer analysis was
performed - did they do this for a representative set of samples as
sanity checks or was this done systematically?
- If a “discovery” dataset is used, has a “validation” cohort been
assessed and/or has the issue of false discovery been addressed?
- Independent confirmation of RNA-seq data in individual fish by
RT-qPCR is lacking.
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?
- Yes.
- 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?
- In general, the work of others was appropriately cited in the
manuscript, although there was consensus that the Discussion could
be strengthened by more clearly describing how this study differs
from the findings of the Altan E, et al ., paper.
- Altan E, Kubiski SV, Boros A, Reuter G, Sadeghi M, Deng X,
Creighton EK, Crim MJ, Delwart E. 2019. A Highly Divergent
Picornavirus Infecting the Gut Epithelia of Zebrafish (Danio
rerio) in Research Institutions Worldwide. Zebrafish16 :291– 299. doi:10.1089/zeb.2018.1710
- 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.
- Overall, the writing was lean and straightforward, and written in a
way to engage broad audience.
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.
- Most important finding, easily understandable by a layperson, was
the actual discovery of ZfPV.
- Likely to improve integrity of future zebrafish infection/innate
immunity studies.
- Highlights method to discover new viruses in existing datasets. This
would be further strengthened by a candid analysis of the limits ofin silico discovery methods.
- 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.
- Yes, there was broad agreement that the current manuscript is a
significant advance without further extension.
Impact: Extensibility (1–4 or N/A scale) SCORE = N/A
- 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)?
- This criterion is only valuable as a scoring parameter if it is
present, indicated by the N/A option if it simply doesn’t apply. 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).
- N/A