PREreview of Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses

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: 

Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses

Jing Wang¹, Yuan-fei Pan², Li-fen Yang³, Wei-hong Yang³, Chu-ming Luo⁴, Juan Wang³,

Guo-peng Kuang³, Wei-chen Wu¹, Qin-yu Gou¹, Gen-yang Xin¹, Bo Li⁵, Huan-le Luo⁴, Yao-qing Chen⁴, Yue-long Shu⁴, Deyin Guo^1,6, Zi-Hou Gao³, Guodong Liang⁷, Jun Li⁸, Edward C. Holmes^9*, Yun Feng^3*, Mang Shi^1*

doi: https://doi.org/10.1101/2022.11.23.517609

We 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: Bats are reservoir hosts for emerging and re-emerging viruses of concern, including influenza viruses and coronaviruses with pandemic potential. Understanding the natural history of these emerging viruses in their reservoir hosts is a top priority for global surveillance efforts. In this manuscript, Wang, J., et al. study the viromes of individual bats from different species collected in Yunnan province to start to understand how viruses of concern co-infect certain bats and/or transmit between species, and to attempt to identify viruses with characteristics that could support zoonotic transmission to humans. This focus on individual bat viromes improves upon previous studies that pooled data from bats of the same species. The authors collected rectal samples from 149 bats from 15 different species from six areas in Yunnan province and harvested RNA for metatranscriptomic sequencing. They focused on viruses that infect mammals. Initial characterization of viromes showed the most frequent viral families across all bat species were Reoviridae, Picornaviridae and Coronaviridae. One third of bats were co-infected, but co-infection rates differed between bat species. Relatively few virus species were detected in more than two bat species. Using a Poisson regression model, they showed that there is a significant correlation between virome composition and the physical proximity and phylogenetic relatedness of bats. To tie their research into potential viral emergence they first identified viruses that are closely related to known human or livestock pathogens. Of these identified “viruses of concern” they focused on several coronaviruses, two of which (BtSY1 and BtSY2) were closely related to SARS-CoVs based upon similarity in RdRp sequences. To further characterize BtSY1 and BtSY2 they compared the sequences of several genes (RdRp, NTD, RBD of spike and N) to other related coronaviruses. From this analysis they showed BtSY1 clusters with the “S-1” clade of coronaviruses (like SARS-CoV) and BtSY2 clusters with the “S-2” clade (like SARS-CoV-2). Comparison of whole genome sequences demonstrated that BtSY1 is 93% identical to human SARS-CoV viruses and BtSY2 is 92% identical to SARS-CoV-2. They also provided evidence for recent recombination events in the origin of BtSY2. Finally, using structural homology-modeling of BtSY2 RBD they hypothesize BtSY2 can use human ACE2 receptor to enter cells like SARS-CoV-2. Overall, these studies suggest that transmission of viruses between different bat species can lead to co-infection of individuals required for recombination events, analogous to the “antigenic shift” events that cause periodic emergence of pandemic influenza viruses.

OVERALL ASSESSMENT: This preprint breaks new ground in the study of viromes in reservoir hosts for the purpose of pandemic surveillance. Study of viromes in individual bats from diverse, geographically linked species were required to get a clear assessment of potential transmission, co-infection and recombination events. Importantly, this approach enabled the discovery of two novel bat viruses closely related to SARS-CoV and SARS-CoV-2, which demonstrates the value of the approach. Comparative genomic analysis revealed recombination events in the origin of BtSY2.

STRENGTHS: A clear strength of this study is the novel approach of investigating viromes of individual bats from different geographically linked species, which allowed the authors to investigate co-infection and potential recombination events. This provides a roadmap for the identification of novel viruses, and virus-host combinations, that increase risk of spillover to humans. The conclusions are generally well supported throughout, and the identification of potential viruses of concern closely related to SARS-CoV and SARS-CoV-2 is undoubtedly very important and will spur future studies.

WEAKNESSES: The primary weaknesses of the study relate to communication. The impact of this study is clear but could be further enhanced by improvements in data presentation and expository writing. We think that it could be more impactful if split into two ‘back-to-back’ manuscripts, with the first focused on the metatranscriptomic studies and cataloguing diverse new viruses discovered and their potential transmission between geographically linked hosts from different bat species, and the second focused on the two novel sarbecoviruses of concern and their properties. This would give the different components of this impactful study the attention they deserve.

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5

·       Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] 

·       Figure 2: We found it challenging and overwhelming to interpret the wealth of information presented in this figure. The top of the heat map has the samples grouped by three different parameters (host genus, host species, and sample location). Potentially limiting it to a single parameter along each axis would aid in making comparisons across the parameters easier.

·       Figure 4A: We were uncertain about what branch length represents in this figure. Please clarify. Could the green circles representing “other viruses” be condensed to a single circle with the number of viruses it represents, or omitted, since the viruses are not named and not central to the message of the figure?

·       Figure 5: We were overwhelmed by the amount of information in this figure. This could be improved by limiting the figure to display the tree of one or two viral families that include the coronaviruses that form the focus for the remainder of the preprint. The other trees could move to the data supplement.

·       Figure 6B: The recombination analysis is fascinating, but underdeveloped, and left us wanting more. The reader would benefit from higher resolution information about proposed sites of recombination (maybe provided by insets?) and how they relate to known recombination hot spots. Undoubtedly, informative comparisons could be made between other related viral genomes. To help the reader make comparisons, colour schemes should be harmonized across the different plots.

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

·       Generally, this was viewed as acceptable throughout.

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

·       Generally, this was viewed as acceptable throughout.

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

·       Generally, this was viewed as acceptable throughout.

·       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. 

·       Was the health of the animals assessed? We discussed how findings might differ between healthy and sick bats, and if this would be reflected in viral load or diversity.    

·       Knowing that bat trapping and sampling was conducted over time made us wonder about how that might affect the genotypes of viruses circulating in these populations over different years. Was this information collected? Is there any evidence that viromes changed over time in certain bat species in the study?  

2. Quality: Completeness (1–3 scale)  SCORE = 2

·       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. 

·       The early exclusion of non-mammalian viruses should be justified.

·       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. 

·       Generally, this was viewed as acceptable throughout.

3. Quality: Reproducibility (1–3 scale) SCORE = 1.5

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

·       N/A

·       Is there sufficient raw data presented to assess rigor of the analysis? 

·       Generally, this was viewed as acceptable throughout.

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

·       Generally acceptable. We thought that providing a clearer rationale for narrowing focus to mammalian viruses, and then the novel sarbecoviruses, would be helpful.  

·       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 (1–4 scale but generally not the basis for acceptance or rejection)  SCORE = 2

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

·       Generally, this was viewed as acceptable throughout.

·       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?

·       Generally, this was viewed as acceptable throughout.

·       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 basis for decisions. 

·       The impact of this study is clear but could be further enhanced by improvements in data presentation and expository writing.

·       We think that it could be more impactful if split into two ‘back-to-back’ manuscripts, with the first focused on the metatranscriptomic studies and cataloguing diverse new viruses discovered and their potential transmission between geographically linked hosts from different bat species, and the second focused on the two novel sarbecoviruses of concern and their properties. This would give the different components of this impactful study the attention they deserve.

MORE SUBJECTIVE CRITERIA (IMPACT) 

1. 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. 

·       The work already represents a substantial advance. A clear strength of this paper is the roadmap to characterizing the viromes of individual reservoir hosts, and the demonstration of co-infection events that provide opportunities for recombination and emergence of novel viruses. Beyond this, the identification and partial characterization of the two novel sarbecoviruses, BtSY1 and BtSY2, represents a very important finding, along with evidence for recombination in the genesis of these viruses.   

2. Impact: Extensibility (1–4 or N/A scale) SCORE = 2.5

·       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)? 

·       There are certainly opportunities to extend these studies to experimental infection models, but this exceeds the reasonable scope of this research. However, we were keen to extend the studies of BtSY2 RBD binding to ACE2, which were limited to in silicoanalyses. Synthesis of BtSY2 Spike protein and testing directly in ACE2 binding assays is quite feasible and straightforward, as is the creation of BtSY2 pseudoviruses that could be used to test virus entry mechanisms and neutralization by animal sera. We view such experiments as achievable and well within the scope of the current study.

·       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

Competing interests

The author declares that they have no competing interests.