Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, 90 ticks from Tibet were sorted into six pools and subjected to high-throughput sequencing, resulting in the detection of viruses belonging to 35 families. Five viral sequences were selected for more detailed genomic and phylogenetic analysis.    

 

MAJOR

 

Early in the Results section, the authors begin by describing viruses from the Chuviridae, Phenuiviridae, and Rhabdoviridae. It would be clearer to first provide an overall summary of all viruses detected before proceeding to detailed descriptions of individual viruses or virus families. For example, an early sentence could state: “A total of [insert number] viruses representing 35 families were identified (Table [insert table number]).” This should be accompanied by a table listing, at minimum, the virus name, genus, family, and GenBank accession number for every virus detected, and possibly the % nucleotide identity with closest known relative. It is also currently unclear how many of the detected viruses were novel versus previously described. Please clarify this early in the Results section by stating the total number of novel viruses and the total number of known viruses identified.

 

The Phenuiviridae sequence is only 3,185 bp and primarily contains L-protein sequence. However, phenuivirus genomes are segmented and total approximately 8.1–25.1 kb so it is not correct to say that a “near complete genome” was sequenced

 

The authors explain that the sequences were deposited into GenBase under accession 570

numbers C_AA501328.1–C_AA501332.1. Is GenBase a typo? Do the authors mean GenBank? If the sequences were not submitted into GenBank, they need to be. It also appears that only five sequences were submitted into the aforementioned database. What about the sequences for the other viruses that were detected?  

 

What tick species were collected? Perhaps this info is provided in the manuscript but if it is, I missed it. If the ticks were not identified according to species, COI sequencing needs to be performed or the tick-derived reads need to be compared to other tick sequences in the GenBank database.    

 

The claims of inter-library heterogeneity are somewhat overinterpreted. The six libraries contain different numbers of pooled ticks (10 to 17 ticks per pool). The manuscript makes fairly strong statements about differences in viral diversity and composition among the libraries which I think need to be omitted from the manuscript.

 

There is considerable repetition between Results and Discussion. The Discussion repeats the same identities, phylogenetic relationships and geographic comparisons already provided in the Results.

 

MINOR

 

Abstract, line 16: replace “and 35 viral families were identified” with “viruses belonging to 35 families were identified”

 

Abstract, lines 17-18: family names (Chuviridae, Phenuiviridae etc.) need to be italicized. Do this elsewhere too

 

After reading the abstract, it is not clear to me if any novel viruses were discovered

 

Intro, line 34: replace “health[1,2]” with “health [1,2]”. Do this elsewhere too -that is, there needs to be a space between the citations and the last word of the sentence

 

Methods, line 76: replace “wild tick samples” with “wild ticks”

 

Sections 2.2 and 2.3 of the methods – several paragraphs are much too short (consisting of only one sentence) and they need to be merged with other paragraphs

 

Methods – in many cases, the city and country of companies (Qiagen, New England BioLabs etc.) are not mentioned

 

Bioinformatics, lines 111: replace “to reduce host-derived sequence contamination” with “to reduce the amount of host-derived sequences” – I don’t think it’s correct to refer to host reads as contamination

 

Line 186: 47.77% - I think that one decimal place is sufficient (same applies elsewhere)

Author Response

Response to Reviewer 1

We thank the reviewer for the constructive comments. We have revised the manuscript and supplementary tables accordingly. The revisions are highlighted in the manuscript, and our point-by-point responses are provided below.

MAJOR COMMENTS

Comment 1:
Early in the Results section, the authors begin by describing viruses from the Chuviridae, Phenuiviridae, and Rhabdoviridae. It would be clearer to first provide an overall summary of all viruses detected before proceeding to detailed descriptions of individual viruses or virus families. For example, an early sentence could state: “A total of [insert number] viruses representing 35 families were identified (Table [insert table number]).” This should be accompanied by a table listing, at minimum, the virus name, genus, family, and GenBank accession number for every virus detected, and possibly the % nucleotide identity with closest known relative. It is also currently unclear how many of the detected viruses were novel versus previously described. Please clarify this early in the Results section by stating the total number of novel viruses and the total number of known viruses identified.

Response:
Thank you for this suggestion. The Results now begin with an overall summary of the sequencing data and viral taxonomic assignments before describing the representative sequences. We report 101 distinct read-based viral taxonomic assignment labels across the six libraries and assignments to 35 viral families.

Supplementary Table S2 provides the complete inventory, including the assignment labels, available genus and family annotations from the original MEGAN taxonomic paths, and representative reference protein accessions from the exported BLASTx matches. Where the source taxonomy did not provide a named genus or family, this is explicitly indicated. Four labels without an eligible representative reference accession have been retained with the accession marked “Not available.”

We distinguish these read-based assignments from the five characterized assemblies. The reference protein accessions document the sequences supporting the BLASTx classifications; they are not study-generated nucleotide accessions. GenBank and GenBase nucleotide accessions for the five assemblies are reported separately in Tables S2 and S3. The other classification labels do not each represent an independently recovered and deposited viral genome.

No novel viruses were identified or proposed in this study, as now stated in the Abstract and early Results. The five characterized assemblies contain regions closely related to three previously reported comparison viruses: Bole Tick Virus 1, Hebei mivirus 1, and Tacheng tick virus 7. We have not equated the 101 classification labels with 101 confirmed viral species, because the inventory includes unclassified and higher-rank labels. Consequently, these assignments do not provide a validated count of distinct known viral species. Nucleotide identity was not added because the retained sequence comparisons were BLASTx amino acid comparisons.

Changes: Sections 2.4 and 3.1; Abstract; Supplementary Tables S2 and S3.

Comment 2:
The Phenuiviridae sequence is only 3,185 bp and primarily contains L-protein sequence. However, phenuivirus genomes are segmented and total approximately 8.1–25.1 kb so it is not correct to say that a “near complete genome” was sequenced.

Response:
We agree. Xizang-S-2-Phenu35739 is now described as a 3,185-bp partial L-region sequence. Recovery of this region does not establish a near-complete L segment or a complete segmented viral genome. We have corrected the terminology throughout the Abstract, Results, Figure 2 legend, Discussion, Conclusions, and Supplementary Table S3. Conserved motifs are treated as annotation evidence, and read coverage is treated as support for the recovered sequence, rather than proof of genome completeness.

Changes: Section 2.5; Section 3.2; Abstract; Figure 2 legend; Discussion; Conclusions; Supplementary Table S3.

Comment 3:
The authors explain that the sequences were deposited into GenBase under accession numbers C_AA501328.1–C_AA501332.1. Is GenBase a typo? Do the authors mean GenBank? If the sequences were not submitted into GenBank, they need to be. It also appears that only five sequences were submitted into the aforementioned database. What about the sequences for the other viruses that were detected?

Response:
GenBase was the database used for the original sequence submission and was not a typographical error. Following the reviewer’s request, the same five representative assembled sequences have also been submitted to GenBank and assigned the following accession numbers:

Xizang-35-4-Chu4909: QB150284
Xizang-S-2-Phenu35739: QB150285
Xizang-35-3-Rha1320: QB150286
Xizang-35-2-Rha3837: QB150287
Xizang-35-2-Chu1189: QB150288

These accessions have been added to the manuscript and Supplementary Tables S2 and S3. The Data Availability Statement distinguishes accession assignment from completion of GenBank processing and public release.

The other entries in the detected-virus inventory originate from taxonomic classification of unassembled reads. They do not each correspond to a characterized assembled sequence recovered in this study. Therefore, they have reference accessions where available, but no study-generated nucleotide accession. We have clarified this distinction in the manuscript and supplementary table notes.

Changes: Data Availability Statement; Results Sections 3.2–3.4; Supplementary Tables S2 and S3.

Comment 4:
What tick species were collected? Perhaps this info is provided in the manuscript but if it is, I missed it. If the ticks were not identified according to species, COI sequencing needs to be performed or the tick-derived reads need to be compared to other tick sequences in the GenBank database.

Response:
Thank you for this comment. The sampling team confirmed that the specimens were collected from vegetation and appeared morphologically similar, but formal individual-level species identification was not performed. We therefore conducted retrospective analysis of tick-derived sequencing reads, following the reviewer’s suggested sequence-comparison approach.

COI screening and assembly recovered nine contigs across the six libraries. These contigs were subsequently compared against the unrestricted NCBI core_nt database using nucleotide BLAST. Representative highest-scoring matches were labelled Rhipicephalus turanicus for Xizang-35-1, Xizang-35-4, and Xizang-22; Hyalomma asiaticum for Xizang-S-2; and Argas persicus for Xizang-35-2 and Xizang-35-3.

The Methods and the Tick_identification worksheet of Supplementary Table S1 provide the screening scope, software settings, reference accessions, nucleotide identities, and query coverage. We report these results as provisional sequence associations at the pooled-library level. They do not establish the species identity of every individual tick or the complete species composition of a pool. We have also removed descriptions implying that all specimens were hard ticks, given the Argas-associated sequences.

Changes: Sections 2.1 and 3.1; Discussion; Supplementary Table S1, Tick_identification worksheet.

Comment 5:
The claims of inter-library heterogeneity are somewhat overinterpreted. The six libraries contain different numbers of pooled ticks (10 to 17 ticks per pool). The manuscript makes fairly strong statements about differences in viral diversity and composition among the libraries which I think need to be omitted from the manuscript.

Response:
We agree and have removed strong interpretations of ecological heterogeneity and claims of statistical or individual-level differences. Each pooled library is now treated as one analytical unit, and the results are presented as descriptive detection patterns and within-library composition.

Supplementary Table S1 reports the pool sizes, sequencing-stage read counts, viral-assigned R1 counts, and corresponding denominators and percentages. The Methods explain the calculation of within-library family proportions. We explicitly acknowledge that relative proportions do not correct for unequal pool sizes or detection sensitivity.

The sampling team clarified that ticks from the same site were combined into one pool. Because there is only one pooled library per sampling site, the design does not provide within-site biological replication. We therefore do not attribute the observed profiles to particular ecological drivers or infer differences in individual-tick infection burden. Taxa detected only in one library are not described as biologically unique to that location or pool.

Changes: Sections 2.2, 2.4, and 3.1; Figure 1 legend; Discussion; Conclusions; Supplementary Table S1.

Comment 6:
There is considerable repetition between Results and Discussion. The Discussion repeats the same identities, phylogenetic relationships and geographic comparisons already provided in the Results.

Response:
The Discussion has been revised to focus on interpretation and limitations. Detailed amino acid identities, aligned regions, reference-protein coverage, and individual phylogenetic relationships are retained in the Results and Supplementary Table S3. The Discussion no longer repeats these numerical comparisons and instead explains the limits of partial sequences, pooled sampling, incomplete specimen metadata, and the absence of independent validation. Geographic explanations are presented as hypotheses rather than established transmission mechanisms.

Changes: Results Sections 3.2–3.4; Discussion; Supplementary Table S3.

MINOR COMMENTS

Comment 1:
Abstract, line 16: replace “and 35 viral families were identified” with “viruses belonging to 35 families were identified”.

Response:
The Abstract has been revised to state: “Viruses belonging to 35 families were identified based on read-level taxonomic assignments.”

Changes: Abstract.

Comment 2:
Abstract, lines 17-18: family names (Chuviridae, Phenuiviridae etc.) need to be italicized. Do this elsewhere too.

Response:
Formal virus family names have been italicized in the Abstract and throughout the manuscript text.

Changes: Abstract and manuscript text.

Comment 3:
After reading the abstract, it is not clear to me if any novel viruses were discovered.

Response:
The Abstract now explicitly states: “No novel viruses were identified in this study.” The Results and Conclusions use the same interpretation.

Changes: Abstract; Section 3.1; Conclusions.

Comment 4:
Intro, line 34: replace “health[1,2]” with “health [1,2]”. Do this elsewhere too—that is, there needs to be a space between the citations and the last word of the sentence.

Response:
We have checked and corrected spacing between the preceding text and numbered citations throughout the manuscript.

Changes: Manuscript text.

Comment 5:
Methods, line 76: replace “wild tick samples” with “wild ticks”.

Response:
The wording “wild tick samples” has been removed. The revised Sample Collection section refers to “90 wild, free-living ticks,” consistent with the confirmed environmental collection method.

Changes: Section 2.1.

Comment 6:
Sections 2.2 and 2.3 of the methods—several paragraphs are much too short (consisting of only one sentence) and they need to be merged with other paragraphs.

Response:
The short method statements have been combined into coherent paragraphs describing sample processing, nucleic acid extraction and library construction, and the computational workflow.

Changes: Sections 2.2 and 2.3.

Comment 7:
Methods—in many cases, the city and country of companies (Qiagen, New England BioLabs etc.) are not mentioned.

Response:
Manufacturer locations have been added, including QIAGEN (Hilden, Germany), Invitrogen (Carlsbad, CA, USA), New England BioLabs (Ipswich, MA, USA), Millipore (Darmstadt, Germany), and Illumina (San Diego, CA, USA).

Changes: Section 2.2.

Comment 8:
Bioinformatics, lines 111: replace “to reduce host-derived sequence contamination” with “to reduce the amount of host-derived sequences”—I don’t think it’s correct to refer to host reads as contamination.

Response:
We agree. The host-depletion description now begins with “To reduce the amount of host-derived sequences.” Host reads are no longer described as contamination in this statement.

Changes: Section 2.3.

Comment 9:
Line 186: 47.77%—I think that one decimal place is sufficient (same applies elsewhere).

Response:
GC content and comparable summary percentages are now displayed to one decimal place. Very small viral-read percentages are retained to three decimal places to avoid rounding low proportions to 0.0%; this exception is explained in the Results and Supplementary Table S1 note. Amino acid identities retain two decimal places to distinguish very high identity from exact identity. The reported precision is therefore specified according to the information represented.

Changes: Sections 2.5 and 3.1; Supplementary Tables S1 and S3.

Reviewer 2 Report

Comments and Suggestions for Authors

• Sampling information and tick identification. The manuscript should provide substantially more information regarding the 90 ticks used in the study. Please specify the sampling localities, collection dates, altitude or elevation range, collection methods, host origin where applicable, tick species, developmental stages, sex distribution, and the criteria used to assign specimens to the six sequencing pools. These variables are essential for interpreting ecological and geographical patterns in the detected virome. If some of these data were not collected, this should be explicitly acknowledged and the corresponding ecological interpretations should be moderated.

• Host sequence depletion. The bioinformatics workflow removes host reads by alignment against the Ixodes scapularis reference genome. Please justify the use of this particular reference genome for Tibetan tick samples and clarify whether the sampled ticks belonged to Ixodes or other genera. If the samples contained different tick species, substantial host derived reads may remain after filtering. The authors should discuss this limitation and, where feasible, consider a broader or species appropriate host depletion strategy.

• Pooling strategy and library comparability. The six pools contain unequal numbers of individual ticks. Please provide the sequencing depth for each library, the number and proportion of viral reads in each library, and the normalization procedure used before comparing viral abundance. Comparisons of relative abundance and viral richness among pools containing different numbers of ticks and potentially different sequencing depths should be interpreted cautiously. The current findings are most appropriately considered descriptive unless statistical comparison and adequate biological replication can be demonstrated.

• Negative controls and contamination filtering. The Methods indicate that blank controls were processed from extraction through sequencing. However, the manuscript does not report what was detected in those controls or how sequences identified in controls were used to remove potential contaminants. Please report the sequencing results from the negative controls and provide the exact thresholds or decision rules used for contaminant exclusion. This is particularly important in viral metagenomic studies where low abundance reagent associated sequences can affect interpretation.

• Definition of complete and near complete genomes. The current definition of a near complete genome as a sequence containing at least one annotatable conserved motif is not sufficiently rigorous. A conserved motif demonstrates the presence of a viral coding region but does not establish genomic completeness. This issue is especially important for the Phenuiviridae sequence, which represents an L protein associated sequence from a segmented viral genome, and for the relatively short Rhabdoviridae sequences compared with the expected genome length of this family. Please distinguish complete genomes, near complete genomes, genomic segments, and partial viral contigs using appropriate criteria. The terminology should then be corrected consistently in the Abstract, Results, figure legends, Discussion, and Conclusions.

• Circular Chuviridae genome. For Xizang 35 4 Chu4909, the manuscript reports a circular genomic topology. Please provide evidence supporting circularity, such as reads spanning the proposed genome junction, mapping coverage across the junction, or another independent assembly validation method. Without such evidence, the sequence should be described as having a putative circular configuration rather than a confirmed circular genome.

• Viral novelty and taxonomic assignment. Several representative sequences show approximately 98.67 to 99.95 percent amino acid identity to previously reported viruses. The authors should therefore clarify whether these sequences represent novel viruses, genetic variants of previously described viruses, or newly documented geographical detections. Terms such as “newly discovered virus” should be avoided unless appropriate ICTV species demarcation criteria and sufficient genomic evidence support such classification. Please state the specific criteria used for assignment to family, genus, subfamily, and, where relevant, species level.

• Bioinformatics reproducibility. Please expand the description of the computational workflow. Information should include relevant database versions and download dates, DIAMOND parameters in addition to the E value threshold, minimum alignment length, sequence identity thresholds, MEGAN lowest common ancestor parameters, criteria for assigning contigs to viral taxa, minimum read or contig thresholds for considering a virus present, sequence coverage, and criteria used after comparison against the non viral database. These details are required to ensure reproducibility.

• Phylogenetic analyses. For each phylogenetic analysis, please report the final alignment length after trimming, the substitution model selected by ModelFinder, the rationale for choosing the reference sequences, the rooting strategy, and the support statistic displayed at each node. The Methods indicate that both SH aLRT and ultrafast bootstrap analyses were performed, whereas the figures appear to show a single support value. Please clarify which statistic is presented. Accession numbers for all sequences included in the phylogenies should also be readily available.

• Interpretation of ecological and geographical patterns. The discussion of geographical connectivity, host associated viral conservation, movement among regions, and possible transport by migratory birds is interesting but remains speculative because tick species, host movement, geographical coordinates, and temporal sampling were not directly incorporated into the analyses. These explanations should be clearly presented as hypotheses rather than inferred transmission mechanisms. The manuscript appropriately acknowledges some of these limitations later in the Discussion, and the same caution should be applied consistently throughout the Results and Discussion.

• Presentation of sequencing and viral sequence data. A supplementary summary table would substantially improve transparency. For each sequencing library, the authors should consider reporting total reads, quality controlled reads, host filtered reads, viral reads, and normalized viral abundance. For each representative viral sequence, the table could include contig length, read depth or coverage, best database match, percentage identity, query coverage, predicted open reading frames, taxonomic assignment, and accession number. These additions would make the reported viral discoveries substantially easier to evaluate.

• Figures and language. The phylogenetic figures should be revised so that taxon names, accession numbers, branch support values, legends, and scale bars remain clearly legible at publication size. The manuscript would also benefit from careful language editing. Statements containing terms such as “clearly”, “definitively”, “stably”, “extremely high”, and “successfully identified” should be replaced with more neutral language where appropriate. Conclusions should remain proportional to what can be established from pooled metagenomic sequence data without viral isolation or individual level validation.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

We thank the reviewer for the detailed and constructive comments. We have expanded the Methods and supplementary tables, corrected genome-completeness terminology, clarified the scope of the sequence evidence, and moderated the ecological and geographical interpretations. We have also revised the language and Conclusions. Changes are highlighted in the manuscript. Our point-by-point responses are provided below.

Comment 1: Sampling information and tick identification

The manuscript should provide substantially more information regarding the 90 ticks used in the study. Please specify the sampling localities, collection dates, altitude or elevation range, collection methods, host origin where applicable, tick species, developmental stages, sex distribution, and the criteria used to assign specimens to the six sequencing pools. These variables are essential for interpreting ecological and geographical patterns in the detected virome. If some of these data were not collected, this should be explicitly acknowledged and the corresponding ecological interpretations should be moderated.

Response:
We have obtained supplementary collection records and clarification from the sampling team. The 90 ticks were collected from vegetation at six sampling sites in Tibet using a cloth-based method recorded as flagging/dragging. No ticks were collected directly from vertebrate hosts.

Bayi District and Gongbujiangda, Lang, Bomi, Motuo, and Chayu counties correspond to Xizang-35-1, Xizang-35-2, Xizang-35-3, Xizang-35-4, Xizang-S-2, and Xizang-22, respectively. Pool allocation was based on sampling site: ticks from the same site were combined into one pool. Equal volumes of supernatant from individually homogenized ticks were used for pooling.

Supplementary Table S1 reports the five available collection dates in July 2026; the Bomi date is marked “Not available.” Supplied coordinates were labelled WGS84, but their provenance and whether they represent exact collection points were not documented. The reported elevations of approximately 1,200–3,410 m are reference estimates rather than documented field measurements. Developmental stage and sex were not recorded.

The collected specimens appeared morphologically similar, but formal individual-level species identification was not performed. We therefore investigated tick taxonomic associations retrospectively using COI sequences recovered from sequencing reads before host depletion. Nine contigs were recovered and compared against the unrestricted NCBI core_nt database. Representative highest-scoring matches were labelled Rhipicephalus turanicus, Hyalomma asiaticum, or Argas persicus, depending on the library. Methods and Supplementary Table S1 provide the screening scope, software settings, reference accessions, nucleotide identities, and query coverage.

These results are reported as provisional pooled-library sequence associations, not identification of every individual tick or the complete species composition of each pool. Similar external appearance was not treated as confirmation that all specimens belonged to one species. Missing metadata and the absence of replicate libraries within sampling sites are acknowledged, and interpretation remains descriptive.

Changes: Sections 2.1, 2.2, and 3.1; Discussion; Supplementary Table S1, Pool_metadata and Tick_identification worksheets.

Comment 2: Host sequence depletion

The bioinformatics workflow removes host reads by alignment against the Ixodes scapularis reference genome. Please justify the use of this particular reference genome for Tibetan tick samples and clarify whether the sampled ticks belonged to Ixodes or other genera. If the samples contained different tick species, substantial host derived reads may remain after filtering. The authors should discuss this limitation and, where feasible, consider a broader or species appropriate host depletion strategy.

Response:
The Ixodes scapularis reference genome, GCF_016920785.2, was available when the original analysis was performed, before specimen-level tick identities had been established. It was used as a general reference for computational subtraction of tick-derived sequences, rather than because all specimens had been confirmed as Ixodes scapularis.

The retrospective COI comparisons suggested Rhipicephalus-, Hyalomma-, and Argas-associated sequences at the pooled-library level. We now explicitly acknowledge that a single Ixodes reference does not represent this inferred taxonomic breadth and may leave residual tick-derived reads or endogenous viral elements.

The subsequent COI analysis did not change the original host-depletion workflow. We do not claim that an additional species-specific or broader host-filtering analysis was performed. This limitation is stated in the Methods and Discussion.

Changes: Section 2.3; Discussion; Supplementary Table S1.

Comment 3: Pooling strategy and library comparability

The six pools contain unequal numbers of individual ticks. Please provide the sequencing depth for each library, the number and proportion of viral reads in each library, and the normalization procedure used before comparing viral abundance. Comparisons of relative abundance and viral richness among pools containing different numbers of ticks and potentially different sequencing depths should be interpreted cautiously. The current findings are most appropriately considered descriptive unless statistical comparison and adequate biological replication can be demonstrated.

Response:
We agree. Supplementary Table S1 now reports raw, post-trimming, and reference-filtered read counts for each library, together with unique viral-assigned R1 counts, their corresponding post-filter R1 denominators, and viral-read percentages.

Across the six libraries, 69,526 unique R1 identifiers were assigned to Viruses or its descendants, representing 0.057% of the 122,649,264 R1 reads retained after both reference-filtering steps. The corresponding per-library counts and percentages are provided in the Read_statistics worksheet.

For the stacked bar plot, each exported family-profile value was divided by the sum of all 35 family-profile values within the same library. These within-library percentages are provided in the Family_proportions worksheet. This denominator is distinct from the total number of post-filter sequencing reads. Because the supplied MEGAN family export contains fractional values, it is not described as an unnormalized raw-read count table.

We have clarified that these transformations describe composition and do not correct for unequal tick numbers or detection sensitivity. Each library represents one site-based pool, and there are no replicate libraries within sites. Comparisons are therefore descriptive; they do not establish statistical ecological differences, individual infection prevalence, or differences in absolute viral burden.

Changes: Sections 2.2, 2.4, and 3.1; Figure 1 legend; Discussion; Conclusions; Supplementary Table S1.

Comment 4: Negative controls and contamination filtering

The Methods indicate that blank controls were processed from extraction through sequencing. However, the manuscript does not report what was detected in those controls or how sequences identified in controls were used to remove potential contaminants. Please report the sequencing results from the negative controls and provide the exact thresholds or decision rules used for contaminant exclusion. This is particularly important in viral metagenomic studies where low abundance reagent associated sequences can affect interpretation.

Response:
Re-examination of the experimental records showed that the original statement about matched blank controls was inaccurate. No batch-matched extraction or library-preparation blanks were included, and no matched negative-control libraries were sequenced for these six tick pools. We have corrected the Methods accordingly.

We therefore do not report negative-control sequencing results or control-derived contaminant-exclusion thresholds. The computational background-reference filter is described separately in Section 2.3 and is not presented as a substitute for experimental negative controls.

The absence of matched controls is explicitly acknowledged in the Discussion and Conclusions. It limits assessment of reagent- or laboratory-associated background, particularly for low-abundance assignments. We cannot retrospectively provide a control dataset that was not generated for this batch, and the biological origins of potentially affected assignments remain unresolved.

Changes: Sections 2.3 and 2.7; Discussion; Conclusions.

Comment 5: Definition of complete and near complete genomes

The current definition of a near complete genome as a sequence containing at least one annotatable conserved motif is not sufficiently rigorous. A conserved motif demonstrates the presence of a viral coding region but does not establish genomic completeness. This issue is especially important for the Phenuiviridae sequence, which represents an L protein associated sequence from a segmented viral genome, and for the relatively short Rhabdoviridae sequences compared with the expected genome length of this family. Please distinguish complete genomes, near complete genomes, genomic segments, and partial viral contigs using appropriate criteria. The terminology should then be corrected consistently in the Abstract, Results, figure legends, Discussion, and Conclusions.

Response:
We agree and have removed the conserved-motif-based definition of a near-complete genome. Conserved motifs are now used as annotation evidence.

Section 2.5 distinguishes completeness terminology according to expected genome length and organization, representation of genomic segments and coding regions, terminal or junction evidence, and read support. For segmented viruses, recovery of one L-associated region does not establish recovery of the complete genome.

None of the five representative assemblies is designated a complete or near-complete viral genome. Phenu35739, Rha3837, and Rha1320 are reported as partial L-region sequences; Chu1189 is a partial genomic sequence; and Chu4909 has a putative circular configuration with unconfirmed topology and genome completeness.

The corresponding terminology has been corrected consistently in the Abstract, Results, figure legends, Discussion, Conclusions, and Supplementary Table S3. Whole-contig read coverage is distinguished from genome completeness.

Changes: Section 2.5; Sections 3.2–3.4; Abstract; Figures 2–4 legends; Discussion; Conclusions; Supplementary Table S3.

Comment 6: Circular Chuviridae genome

For Xizang 35 4 Chu4909, the manuscript reports a circular genomic topology. Please provide evidence supporting circularity, such as reads spanning the proposed genome junction, mapping coverage across the junction, or another independent assembly validation method. Without such evidence, the sequence should be described as having a putative circular configuration rather than a confirmed circular genome.

Response:
We inspected read mapping to the 11,234-bp Chu4909 assembly. Mapping yielded 29,131 individual reads, a mean depth of 369.1×, and 100.0% read-coverage breadth across the assembled reference.

However, the inspected potential junction-crossing alignments had insufficient flanking alignment or substantial mismatches and did not provide reliable support for the proposed end-to-start junction. The 9-bp repeated sequence used during manual joining, and setting the mapping reference to circular, were not treated as independent evidence of circular topology.

We therefore consistently describe a putative circular configuration and explicitly state that topology and genome completeness remain unconfirmed. Coverage across the assembled sequence is not used as a substitute for junction validation.

Changes: Section 2.5; Section 3.3; Abstract; Figure 3 legend; Discussion; Conclusions; Supplementary Table S3.

Comment 7: Viral novelty and taxonomic assignment

Several representative sequences show approximately 98.67 to 99.95 percent amino acid identity to previously reported viruses. The authors should therefore clarify whether these sequences represent novel viruses, genetic variants of previously described viruses, or newly documented geographical detections. Terms such as “newly discovered virus” should be avoided unless appropriate ICTV species demarcation criteria and sufficient genomic evidence support such classification. Please state the specific criteria used for assignment to family, genus, subfamily, and, where relevant, species level.

Response:
No novel virus, new species, new strain, or independently established geographic variant is claimed. The five assemblies are described as sequence detections in Tibetan tick pools that are closely related to previously reported viruses. Their amino acid identities refer to the compared L-protein regions, not whole-genome nucleotide identities.

Read-level assignments follow the original MEGAN taxonomy and recorded LCA settings. For the representative assemblies, family-level annotations and genus- or subfamily-related associations are supported by protein homology and comparisons with selected reference sequences. These associations are distinguished from formal species assignment.

The Results describe the relevant ICTV criteria for the compared groups, including the Uukuvirus L-protein identity criterion and the Mivirus criterion based on complete L proteins. Partial-region identity and tree placement are not used to assign new species. The Rhabdoviridae sequences are reported as partial L-region detections with a tentative phylogenetic association near Deltarhabdovirinae references; no formal genus, species, strain, or variant designation is assigned.

Read-based inventory entries are called viral taxa or taxonomic assignment labels, with unclassified and higher-rank labels retained as such. The Abstract and early Results explicitly state that no novel viruses were identified.

Changes: Sections 2.3–2.5 and 3.1–3.4; Abstract; Discussion; Conclusions; Supplementary Tables S2 and S3.

Comment 8: Bioinformatics reproducibility

Please expand the description of the computational workflow. Information should include relevant database versions and download dates, DIAMOND parameters in addition to the E value threshold, minimum alignment length, sequence identity thresholds, MEGAN lowest common ancestor parameters, criteria for assigning contigs to viral taxa, minimum read or contig thresholds for considering a virus present, sequence coverage, and criteria used after comparison against the non viral database. These details are required to ensure reproducibility.

Response:
We have expanded the computational Methods using the available scripts, reports, and confirmed database records.

Section 2.3 reports DIAMOND v2.1.13 and the default E-value threshold of 0.001. The contig search used DAA output (-f 100), up to 25 target sequences per query (-k 25), and 18 threads (-p 18), without additional explicitly specified minimum alignment-length or percentage-identity filters. The local viral database, 2025-9-virus.dmnd, was downloaded on 9 May 2026; nr250731.dmnd was downloaded on 31 July 2025; and the MEGAN mapping database, megan-nr-r2.mdb, was downloaded on 25 June 2025. Separate release or build identifiers were not documented and are not inferred from filenames.

We report the MEGAN Naive LCA settings: minimum bit score 50, maximum E-value 0.01, top percent 10%, minimum support percentage 0.01%, minimum support setting 1, and readCount assignment mode, with paired-read handling disabled. Recorded identity, length, and coverage filters set to zero are described as not additionally imposed, rather than validated detection cutoffs.

Assembled contigs were selected and exported from specific viral-family nodes in MEGAN. For the descriptive UpSet analysis, positive exported assignment values (>0) were scored as detected. This operational rule does not validate species identity or infection.

The retained contig scripts document viral-protein searching followed by MEGAN classification. A separate competitive NVNR screening procedure, viral-versus-nonviral bit-score comparison, equal-score handling rule, or additional competitive exclusion threshold could not be verified. We have removed unsupported claims that such filtering was demonstrated. The unrestricted nr search described in the revised Methods applies to read classification, not demonstrated competitive filtering of assembled contigs.

Sequence-specific mapping settings and coverage definitions are provided in Section 2.5 and Supplementary Table S3. The limitations arising from unavailable database build identifiers and incomplete original records are acknowledged in the Discussion.

Changes: Sections 2.3–2.5; Discussion; Supplementary Tables S1–S3.

Comment 9: Phylogenetic analyses

For each phylogenetic analysis, please report the final alignment length after trimming, the substitution model selected by ModelFinder, the rationale for choosing the reference sequences, the rooting strategy, and the support statistic displayed at each node. The Methods indicate that both SH aLRT and ultrafast bootstrap analyses were performed, whereas the figures appear to show a single support value. Please clarify which statistic is presented. Accession numbers for all sequences included in the phylogenies should also be readily available.

Response:
Section 2.6 and Supplementary Table S4 now report the final sequence counts, alignment lengths after gap filtering, selected substitution models, rooting references, and support statistics for all three analyses:

Figure 2: 37 sequences, 4,738 amino-acid positions, model Q.pfam+F+R6.
Figure 3: 24 sequences, 2,336 amino-acid positions, model LG+F+I+R4.
Figure 4: 31 sequences, 2,491 amino-acid positions, model rtREV+F+R5.

The reference-selection rationale is stated: closely related tick-associated viral L proteins and representative homologous proteins from related groups were included for sequence comparison. The set was not designed as a systematic geographical sample.

The displayed trees were rooted on the branches separating Abras virus, Wenling fish chu-like virus, and the Gammarhabdovirinae reference clade from the remaining sequences in Figures 2, 3, and 4, respectively.

Support was assessed using 1,000 ultrafast bootstrap replicates with NNI optimization. Single values displayed at nodes are UFBoot support percentages; SH-aLRT values are not displayed. The Methods and figure legends have been corrected to clarify this correspondence.

Supplementary Table S5 lists all reference protein accessions, including those used for rooting. The study-generated nucleotide accessions are provided in Supplementary Table S3.

Changes: Section 2.6; Figures 2–4 legends; Supplementary Tables S3–S5.

Comment 10: Interpretation of ecological and geographical patterns

The discussion of geographical connectivity, host associated viral conservation, movement among regions, and possible transport by migratory birds is interesting but remains speculative because tick species, host movement, geographical coordinates, and temporal sampling were not directly incorporated into the analyses. These explanations should be clearly presented as hypotheses rather than inferred transmission mechanisms. The manuscript appropriately acknowledges some of these limitations later in the Discussion, and the same caution should be applied consistently throughout the Results and Discussion.

Response:
We agree and have removed statements implying that high sequence identity or phylogenetic proximity demonstrates geographical movement, host-associated conservation, tick dispersal, or a transmission route.

The Results now describe closely related sequences detected in geographically separated regions. They do not establish widespread distribution or occurrence in intervening locations. Limited and geographically uneven reference sampling is explicitly acknowledged.

The Discussion states that host movement, tick dispersal, geographical connectivity, and transport by migratory birds were not measured. Their possible contributions remain hypotheses for future investigation. The newly obtained sampling metadata do not provide the replication or direct movement analyses required to establish these mechanisms.

The same caution is applied consistently in the Results, Discussion, and Conclusions.

Changes: Sections 3.2–3.4; Discussion; Conclusions.

Comment 11: Presentation of sequencing and viral sequence data

A supplementary summary table would substantially improve transparency. For each sequencing library, the authors should consider reporting total reads, quality controlled reads, host filtered reads, viral reads, and normalized viral abundance. For each representative viral sequence, the table could include contig length, read depth or coverage, best database match, percentage identity, query coverage, predicted open reading frames, taxonomic assignment, and accession number. These additions would make the reported viral discoveries substantially easier to evaluate.

Response:
Supplementary Table S1 now provides per-library raw, post-trimming, and reference-filtered read counts, viral-assigned R1 counts, corresponding denominators and percentages, and within-library family proportions.

Supplementary Table S3 provides the five representative sequence lengths, source pools, predicted coding-region summaries, taxonomic associations, study-generated accessions, comparison references, amino acid identities, identical/aligned amino acid counts, reference-protein coverage, BLASTx query coverage, mapped reads, mean depth, and read-coverage breadth. Mapping statistics are included for all five sequences.

Query coverage is the rounded NCBI display value for the submitted L-region nucleotide query; it does not represent coverage of the entire assembled contig or viral genome. Reference-protein coverage and read-coverage breadth are reported separately.

We retain the heading “Compared reference” rather than “Best database match,” because complete hit lists were unavailable to verify a universally best match for every comparison.

The counting scopes are also clarified. S3 mapping counts include individual raw R1 and R2 sequences mapped as unpaired reads, whereas S1 viral taxonomic counts are unique post-filter R1 identifiers. These quantities were not divided by one another or expected to be equal.

Changes: Sections 2.5 and 3.1–3.4; Supplementary Tables S1 and S3 and their notes.

Comment 12: Figures and language

The phylogenetic figures should be revised so that taxon names, accession numbers, branch support values, legends, and scale bars remain clearly legible at publication size. The manuscript would also benefit from careful language editing. Statements containing terms such as “clearly”, “definitively”, “stably”, “extremely high”, and “successfully identified” should be replaced with more neutral language where appropriate. Conclusions should remain proportional to what can be established from pooled metagenomic sequence data without viral isolation or individual level validation.

Response:
We have reviewed the figure labels, reference accession labels, taxonomic-rank legends, and scale-bar descriptions. Coding-region labels distinguish predicted sequence regions from complete genomes. The figure legends explicitly identify UFBoot support percentages, the scale-bar meaning, and the study sequences. All reference accessions are also available in Supplementary Table S5.

The manuscript has been revised to use more neutral, descriptive language. Claims of strong ecological heterogeneity, confirmed circular topology, new viral species or strains, active infection, and viral replication have been removed or qualified according to the available evidence.

The Conclusions have been rewritten to focus on descriptive sequence diversity and phylogenetic relationships. Pooling, unequal sequencing depths, incomplete specimen metadata, absent matched negative controls, and the lack of PCR/RT-PCR, isolation, or individual-level validation are acknowledged. The conclusions do not infer unmeasured infection patterns, ecological drivers, or transmission processes.

Changes: Figures 1–4 and legends; Results; Discussion; Conclusions; Supplementary Tables S3–S5.

ADDITIONAL CLARIFICATIONS ADDRESSING THE ANNOTATED MANUSCRIPT

1. Correspondence line

The duplicated Correspondence label has been removed. The author-information block now contains a single correspondence entry.

Changes: Author-information block.

2. ORF annotation settings

The 400-bp minimum length and ATG start requirement are described as operational settings used in the Geneious ORF screen, not universal biological criteria for viral proteins or genome completeness. The Methods acknowledge that shorter proteins, alternative initiation codons, and truncated coding regions may be missed. Failure to detect an ORF under these settings does not establish absence of a coding region. Homology-supported regions are reported as partial coding regions where completeness was not established.

Changes: Section 2.5; Supplementary Table S3.

3. Mapping, pairing, consensus, and terminal support

Section 2.5 reports Geneious Prime v2024.0.5, the Geneious mapper, the Low Sensitivity / Fastest preset, and the recorded detailed settings. Fine Tuning was None, Do not trim was selected, and R1/R2 sequences were mapped as unpaired reads. Mapped-read counts therefore refer to individual sequences, not read pairs or independent fragments.

No new mapping-derived consensus was exported, and no minimum-depth or allele-frequency rule was applied to generate a replacement consensus. Apart from joining Chu4909 and retaining one copy of the repeated sequence, the final assemblies were not manually altered. The five final submitted sequences contain no N bases or other nucleotide ambiguity codes. Terminal coverage was not assessed separately, and overall coverage breadth does not validate genome termini or circular junctions. These limitations are stated rather than assigned unverified thresholds.

Changes: Section 2.5; Discussion; Supplementary Table S3 note.

4. DNA/RNA recovery and biological sources

The same nucleic-acid extract was used for reverse transcription and subsequent synthesis without post-extraction DNA depletion. Co-extracted DNA and RNA-derived cDNA could therefore contribute to the DNA library; their relative recovery efficiencies were not determined.

The Discussion distinguishes detection in tick-derived pools from infection or replication in ticks. DNA-virus and bacteriophage assignments may reflect blood meals, associated microorganisms, environmental material, or processing-related background. Retrovirus-related assignments are displayed under the reporting label “Endogenous retroviruses (ERVs)” in Figure 1; this category is not independent evidence of genomic integration or viral replication.

Changes: Section 2.2; Discussion; Figure 1.

5. Raw-read archive and library metadata

Supplementary Table S1 links all six specimen libraries to BioProject PRJCA074065, GSA CRA049867, BioSamples SAMC8505619–SAMC8505624, and runs CRR3542369–CRR3542374. The first sequencing-statistics stage represents the original raw R1+R2 data received from the sequencing provider, distinguished from the subsequent trimmed and reference-filtered stages. No matched negative-control libraries were sequenced for this batch, so no such control runs are reported.

Changes: Data Availability Statement; Supplementary Table S1.

6. Collection and ethics framework

The sampling team confirmed that only free-living environmental ticks were collected. No vertebrate animals were captured, restrained, maintained, or dissected, and no ticks were removed directly from vertebrate hosts. Permissions relating specifically to host-animal tick sampling therefore do not apply to these procedures.

Environmental collection was included in the ethics review framework of a National Key R&D Program project on disease prevention and control in plateau animals, under approval No. JSDX20251211001. The Institutional Review Board Statement has been revised to describe this framework. Gongga of Tibet Agricultural and Animal Husbandry University is explicitly acknowledged for assistance with collection.

Changes: Section 2.1; Institutional Review Board Statement; Acknowledgments.

Reviewer 3 Report

Comments and Suggestions for Authors

【Over view】

Hard ticks inhabit a wide range of environmental conditions across the globe. The Qinghai-Tibet Plateau, the region investigated in this study, is characterized by the low temperatures and low oxygen levels typical of the Tibetan Plateau; however, the Virome of the hard ticks inhabiting this area has remained unknown. Consequently, the authors surveyed the viral communities of 90 wild ticks collected in Tibet. The results revealed high viral diversity, with single-stranded RNA (ssRNA) viruses belonging to Phenuiviridae, Chuviridae, and Rhabdoviridae, predominating. Genetic analysis on these viruses, showed that these viruses share a high degree of similarity with tick-associated viruses previously reported in Xinjiang, China, and other regions. The findings of this study are highly significant, as they provide foundational data for future research into the geographical distribution, genetic diversity, and ecological characteristics of RNA viruses harbored by hard ticks in the high-altitude ecosystem of the Qinghai-Tibet Plateau.

 

[Major point]

There are no specific comments, but I would have liked to know if there was any information regarding tick-borne infectious diseases—particularly those caused by RNA viruses—affecting animals or humans in the study area.

 

[Minor point]

None.

Author Response

Response to Reviewer 3

We thank the reviewer for the positive assessment of our manuscript and the helpful suggestion to include additional information on tick-borne viral diseases in the study region.

Comment:
There are no specific comments, but I would have liked to know if there was any information regarding tick-borne infectious diseases—particularly those caused by RNA viruses—affecting animals or humans in the study area.

Response:
Thank you for this suggestion. We have expanded the Discussion to provide relevant regional information on tick-associated RNA viruses. Specifically, we now cite the detection of a proposed Himalayan subtype of tick-borne encephalitis virus in Himalayan marmots in Qinghai Province on the Qinghai–Tibet Plateau (Dai et al., 2018; reference 46), and the identification of Gagosa Mountain virus in Ornithodoros lahorensis ticks collected from sheep in Shigatse, Tibet (Tu et al., 2025; reference 47). We distinguish the report from Qinghai Province, which provides broader plateau context, from the report originating in Tibet.

We also clarify that detection of viral sequences in animals or ticks does not, by itself, demonstrate clinical disease in humans or livestock. The present study did not investigate clinical cases, serological evidence, or disease outcomes and therefore cannot estimate the local burden of tick-borne viral disease. This distinction has been added to ensure that the regional background and the implications of our findings are presented cautiously.

Changes:
Discussion, paragraph beginning “Sequence surveys can inform further investigation of tick-associated viruses”; references 46 and 47.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No concerns

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for their comprehensive responses to my previous comments and for the substantial improvements made to the revised manuscript. The revisions have significantly strengthened the methodological transparency, scientific accuracy, and overall presentation of the study.

The authors have adequately addressed most of the concerns raised during the initial review, particularly regarding sampling information, tick identification, sequencing statistics, genome completeness, phylogenetic analyses, and interpretation of viral diversity. The inclusion of supplementary tables further improves the transparency and reproducibility of the study.

I particularly appreciate the clarification concerning the absence of matched negative controls, the limitations of host sequence depletion, and the revised terminology distinguishing partial viral sequences from complete genomes. The authors have also appropriately moderated claims regarding viral novelty, geographical distribution, infection, and transmission.

Although some methodological limitations remain, these are now explicitly acknowledged and do not necessarily invalidate the descriptive findings presented in the manuscript.

I recommend that the authors undertake a final review to ensure consistency between the manuscript, figures, and supplementary tables, particularly regarding provisional viral taxonomic assignments and tick species identification.

Overall, I am satisfied with the authors' responses and consider the manuscript substantially improved. I have no further major scientific concerns and recommend acceptance.

Comments on the Quality of English Language

The English language has been substantially improved in the revised manuscript. The scientific terminology is generally appropriate, and the Methods, Results, and Discussion are presented clearly and coherently.

The authors have successfully moderated previously overstated interpretations and improved the precision of the scientific language. Minor proofreading is recommended to address any remaining grammatical, punctuation, and formatting inconsistencies before publication.

Overall, the quality of English is satisfactory for scientific publication.