Development of a Quadruplex RT-qPCR for the Detection of Feline Kobuvirus, Feline Astrovirus, Feline Bufavirus, and Feline Rotavirus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

 

 

Comments:

1.    Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 

2.     Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

 

3.    It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

4.     Line 288-289: it would be helpful if the authors would define “PRV”.

Author Response

The Cover Letter

September 30, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the suggestions of the editor and the reviewers. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

  Comments:

1. Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 Response: According to the results of our test, the designed primers and probes could specifically detect the target genes. The fluorescence signals could obtain only from the four viruses and generate amplification curves, and no fluorescence signal could detect from other control viruses and negative controls. No cross-reactivity among the 4 targets within assays was observed, and no bleed-through fluorescence signal from neighboring channels occurred.

2. Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

Response: We agree to the reviewer’s suggestion. The genome sequences of FeKoV, FeAstV, FeBuV, and FRV were downloaded from the NCBI GenBank, and the multiple sequence alignment was performed. The conserved regions were selected to design specific primers and probes. Figure 1 shows the nucleotide sequence alignments of the partial FeKoV VP1 gene, FeAstV ORF2 gene, FeBuV VP2 gene, and FRV NSP4 gene, and the locations of the specific primers and probes designed for the quadruplex RT-qPCR. Please see Figure 1 in the revised manuscript.

In addition, the sequences of the designed primers and probes were performed BLAST analysis in the NCBI GenBank to confirm their specificity. Please see Lines 120-122 in the revised manuscript.

3. It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

Response: Besides FeKoV, FeAstV, FeBuV, and FRV, other viruses such as feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), and feline leukemia virus (FeLV) can also cause gastroenteritis showing clinical signs of vomiting, diarrhea, and dehydration. Their accurate diagnosis depends on the detection of pathogens in laboratory. In our previous report, a triplex quadruplex RT-qPCR was developed for the detection and differentiation of FPV, FCoV, and FeLV.  Therefore, the purpose of this study was to develop a quadruplex RT-qPCR for the detection and differentiation of FeKoV, FeAstV, FeBuV, and FRV. Please see Lines 334-337 in the revised manuscript.

The reference is as follows:

He M, Feng S, Shi K, Shi Y, Long F, Yin Y, Li Z. One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus. Vet World. 2024, 17(5):946-955.

4. Line 288-289: it would be helpful if the authors would define “PRV”.

Response: This is a spelling error. “PRV” should be “FRV”. It has been corrected. Please Lines 321 and 322 in the revised manuscript.

 

Reviewer #2

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 Major concerns

1. Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay.

Response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluated the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validated the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay.

 FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

2. Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions?

Response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

3. Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance.

Response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

4. Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

Major comments

5. Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity.

Response: In Figure 3, only one concentration of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV was used as positive controls, so the standard curves could not generate and show. In addition, no melting curve is generated in RT-qPCR/qPCR using specific TaqMan probe, so we cannot provide it here.

6. Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent.

Response: We agree to the reviewer’s suggestion. The sequences used for comparison to design the specific primers and probes were showed in figure 1, and the information of the virus strains including collected year were showed in Supplementary Table S1-S4. The accessed date of these sequences has been added in the revised manuscript. Please see Figure 1, Supplementary Table S1-S4, and Lines 112-123 in the revised manuscript.

7. Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation.

Response: We agree to the reviewer’s suggestion. The test results of 1869 clinical samples were showed in Table 5. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diseased animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738), and 1.08% (8/738), respectively, while the positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131), and 0.53% (6/1131), respectively. The positivity rates in diseased animals were higher than those in subclinical animals. Please see Lines 290-296 in the revised manuscript.

Minor comments

8. Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality.

Response: We agree to the reviewer’s suggestion. The Times New Roman was used in all figures, and the font sizes were different according to the size of the original images. In addition, Figure 2 and Figure 3 have been revised to improve the quality of the figures. Please see Figure 1 to figure 4 in the revised manuscript.

9. Minor comment 1: Why are sections 2.2 and 2.8 separate?

Response: Section 2.2 belongs to “Materials”, and Section 2.8 belongs to “Methods”. Please see the revised manuscript.

10. Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

Response: We agree to the reviewer’s suggestion. The caption has been added to Figure 2. In Figure 2, the horizontal axis represents the number of Ct values (cycles) and the vertical axis represents the number of template (copies), and their quantities are indicated in the figures. Please see Figure 2 in the revised manuscript.

Minor comments 3 (textual/narrative):

11. Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 13 in the revised manuscript.

12. Line 14: ..The established quadruplex RT-qPCR 'assay'...

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 15 in the revised manuscript.

13. Line 15: Use "reproducibility" instead of "repeatability"

Response: We agree to the reviewer’s suggestion. The "repeatability" has been replaced by "reproducibility" in the revised manuscript. Please see Lines 15, 192, 193, 281, 284, 287, 288, and 357 in the revised manuscript.

14. Line 40, 48, 57, 62: ..genome 'size'..

Response: We agree to the reviewer’s suggestion. The “size” has been added in the revised manuscript. Please see Lines 40, 49, 58, and 63 in the revised manuscript.

15. Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Response:  We agree to the reviewer’s suggestion. The "uneasy contamination/pollution" has been revised in the revised manuscript. Please see Lines 79, and 342 in the revised manuscript.

16. Line 85: ...and also obtains 'provides'.

Response: We agree to the reviewer’s suggestion. The “provides” has been added in the revised manuscript. Please see Lines 87 in the revised manuscript.

17. Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Response: We agree to the reviewer’s suggestion. The target genes were relatively conserved. The references have been cited in the revised manuscript. Please see Lines 120 in the revised manuscript.

18. Line 283-285: Cats pets are important companion.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 316 in the revised manuscript.

19. Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 322-323 in the revised manuscript.

20. Line 337: (make a new paragraph) In addition, co-infections.

Response: We agree to the reviewer’s suggestion. A new paragraph has been added in the revised manuscript. Please see Lines 403 in the revised manuscript.

21. Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.

Response: We agree to the reviewer’s suggestion. According to the reviewer’s suggestion (see the following suggestion), this sentence has been deleted. Please see Lines 443 in the revised manuscript.

22. Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Response: We agree to the reviewer’s suggestion. The sentences have been deleted in the revised manuscript. Please see Lines 441-445 in the revised manuscript.

23. Line 383-384: Uneven font sizes.

Response: We agree to the reviewer’s suggestion. The font size of this sentence has been corrected. Please see Lines 458-460 in the revised manuscript.

Comments on the Quality of English Language

24. Proof-reading is recommended.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of English language. Please see the revised manuscript.

 

Reviewer #3

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR.

1. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions.

Response: The purpose of RT-qPCR is to detect nucleic acids (RNA or DNA). Each technology has its function, and advantages, and also has its limitations. The RT-qPCR can detect viruses at RNA/DNA level, but not at protein level. To detect viruses at protein level, we need other technology and methods. This is beyond the scope of this study.

2. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

Response: The RT-qPCR can detect the target viruses at RNA/DNA level according to the principle of this technique. The purpose of this study is to develop a quadruplex RT-qPCR to detect FeKoV, FeAstV, FeBuV, and FRV. In this study, we did not detect viruses at protein level using other technology such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

3. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Response: In this study, we confirmed that FeBuV existed in Guangxi province using the developed quadruplex RT-qPCR to detect FeBuV in clinical samples. Now, we are trying to isolate and identify this virus in our laboratory.

 

Reviewer #4

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

Response: We agree to the reviewer’s suggestion. The prominent advantage of the developed RT-qPCR has been discussed in the Discussion part. Please see Lines 342-345 and 368-371 in the revised manuscript.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

Response: We agree to the reviewer’s suggestion. The clinical samples from Guangxi province in southern China were used to validate the application of the developed quadruplex RT-qPCR. This does not mean that the primers and probes have geographic limitation. The specific primers and probes were designed based on the genome sequences of FeKoV, FeAstV, FeBuV, and FRV which came from different countries (Supplementary Table S1-S4). The multiple sequence alignment was performed, and the conserved regions were selected to design specific primers and probes. They are suitable for the detection of the strains from different countries around the world. This has been discussed in the Discussion part. Please see Lines 371-380 in the revised manuscript.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

Response: We agree to the reviewer’s suggestion. The meaning of the developed assay has been discussed in the Discussion part. Please see Lines 381-385 in the revised manuscript.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

Response: We agree to the reviewer’s suggestion. The public health significance of the findings of these viruses has been discussed in the Discussion part in the revised part. Please see Lines 433-440 in the revised manuscript.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Response: We agree to the reviewer’s suggestion. The criteria for sample selection have been added in 2.2 Clinical Samples of the revised manuscript. Please see Lines 106-110 in the revised manuscript.

Minor Concerns:

6. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

Response: We agree to the reviewer’s suggestion. The definitions of "probit regression" and "coefficient variation" have been described in the revised manuscript. Please see Lines 188-191 and 197-200  in the revised manuscript.

7. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

Response: We agree to the reviewer’s suggestion. The tables have been checked, and used uniform formatting. The formatting will be corrected by the editor. Please see the revised manuscript.

8. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

Response: We agree to the reviewer’s suggestion. The references have been revised carefully, and the recent references have been updated. Please see the references in the revised manuscript.

9. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

Response: We agree to the reviewer’s suggestion. The section of the design of primers and probes has been revised. Please see Lines 112-125 in the revised manuscript.

10. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

Response: We agree to the reviewer’s suggestion. The workflow of the RT-qPCR development has been summarized and discussed in the Discussion part. Please see Lines 350-354 in the revised manuscript.

 Comments on the Quality of English Language

11. The language should be improved.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of the manuscript. Please see the revised manuscript.

 

Reviewer #5

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1869 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1. Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

Response: We agree to the reviewer’s suggestion. The four viruses can infect cats of all ages, but no vaccine is available for them now. This information has been added in the Introduction part of the revised manuscript. Please see Lines 71-75 in the revised manuscript.

2. What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

Response: We agree to the reviewer’s suggestion. All the samples were collected from subclinical or diseased cats. The feces, and anal/nasal swabs were collected from each cat, and mixed together for detection of FeKoV, FeAstV, FeBuV, and FRV. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869) and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diarrhea animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738) and 1.08% (8/738), respectively. The positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131) and 0.53% (6/1131), respectively. Please see Lines 291-296 in the revised manuscript.

3. Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]”. Why virus was associated with virus gene here?

Response: We agree to the reviewer’s suggestion. This sentence has been deleted. Please see Lines 58-59 in the revised manuscript.

4. “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative.

Response: We agree to the reviewer’s suggestion. “Vaccine Strains” has been changed to “Reference strains”. Please see Line 95 in the revised manuscript.

5. Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

Response: The amplification efficiency of real-time PCR is usually between 90%-110%, and the amplification efficiency of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were 109.798%, 109.965%, 108.551%, and 107.160%, respectively. They are normal values. Please see Lines 243-244 in the revised manuscript.

6. The font size of the last sentence of the main text looks smaller than the others.

Response: Sure. The font size has been corrected. Please see Lines 458-460 in the revised manuscript.

7. In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Response: We agree to the reviewer’s suggestion. “Date” has been substituted with “Year”. The Host column in the tables has been deleted. Please see the Supplementary Table S1-S4 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 

Major concerns

 

Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay. 

 

FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples. 

 

Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions? 

 

Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance. 

 

Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included. 

 

Major comments

 

Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity. 

 

Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent. 

 

Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation. 

 

Minor comments

 

Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality. 

 

Minor comment 1: Why are sections 2.2 and 2.8 separate? 

 

Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

 

Minor comments 3 (textual/narrative): 

 

Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Line 14: ..The established quadruplex RT-qPCR 'assay'...

Line 15: Use "reproducibility" instead of "repeatability"

Line 40, 48, 57, 62: ..genome 'size'..

Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Line 85: ...and also obtains 'provides'..

Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Line 283-285: Cats pets are important companion..

Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Line 337: (make a new paragraph) In addition, co-infections..

Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.  

Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Line 383-384: Uneven font sizes.

Comments on the Quality of English Language Proof-reading is recommended. 

Author Response

The Cover Letter

September 30, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the suggestions of the editor and the reviewers. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

  Comments:

1. Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 Response: According to the results of our test, the designed primers and probes could specifically detect the target genes. The fluorescence signals could obtain only from the four viruses and generate amplification curves, and no fluorescence signal could detect from other control viruses and negative controls. No cross-reactivity among the 4 targets within assays was observed, and no bleed-through fluorescence signal from neighboring channels occurred.

2. Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

Response: We agree to the reviewer’s suggestion. The genome sequences of FeKoV, FeAstV, FeBuV, and FRV were downloaded from the NCBI GenBank, and the multiple sequence alignment was performed. The conserved regions were selected to design specific primers and probes. Figure 1 shows the nucleotide sequence alignments of the partial FeKoV VP1 gene, FeAstV ORF2 gene, FeBuV VP2 gene, and FRV NSP4 gene, and the locations of the specific primers and probes designed for the quadruplex RT-qPCR. Please see Figure 1 in the revised manuscript.

In addition, the sequences of the designed primers and probes were performed BLAST analysis in the NCBI GenBank to confirm their specificity. Please see Lines 120-122 in the revised manuscript.

3. It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

Response: Besides FeKoV, FeAstV, FeBuV, and FRV, other viruses such as feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), and feline leukemia virus (FeLV) can also cause gastroenteritis showing clinical signs of vomiting, diarrhea, and dehydration. Their accurate diagnosis depends on the detection of pathogens in laboratory. In our previous report, a triplex quadruplex RT-qPCR was developed for the detection and differentiation of FPV, FCoV, and FeLV.  Therefore, the purpose of this study was to develop a quadruplex RT-qPCR for the detection and differentiation of FeKoV, FeAstV, FeBuV, and FRV. Please see Lines 334-337 in the revised manuscript.

The reference is as follows:

He M, Feng S, Shi K, Shi Y, Long F, Yin Y, Li Z. One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus. Vet World. 2024, 17(5):946-955.

4. Line 288-289: it would be helpful if the authors would define “PRV”.

Response: This is a spelling error. “PRV” should be “FRV”. It has been corrected. Please Lines 321 and 322 in the revised manuscript.

 

Reviewer #2

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 Major concerns

1. Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay.

Response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluated the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validated the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay.

 FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

2. Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions?

Response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

3. Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance.

Response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

4. Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

Major comments

5. Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity.

Response: In Figure 3, only one concentration of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV was used as positive controls, so the standard curves could not generate and show. In addition, no melting curve is generated in RT-qPCR/qPCR using specific TaqMan probe, so we cannot provide it here.

6. Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent.

Response: We agree to the reviewer’s suggestion. The sequences used for comparison to design the specific primers and probes were showed in figure 1, and the information of the virus strains including collected year were showed in Supplementary Table S1-S4. The accessed date of these sequences has been added in the revised manuscript. Please see Figure 1, Supplementary Table S1-S4, and Lines 112-123 in the revised manuscript.

7. Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation.

Response: We agree to the reviewer’s suggestion. The test results of 1869 clinical samples were showed in Table 5. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diseased animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738), and 1.08% (8/738), respectively, while the positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131), and 0.53% (6/1131), respectively. The positivity rates in diseased animals were higher than those in subclinical animals. Please see Lines 290-296 in the revised manuscript.

Minor comments

8. Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality.

Response: We agree to the reviewer’s suggestion. The Times New Roman was used in all figures, and the font sizes were different according to the size of the original images. In addition, Figure 2 and Figure 3 have been revised to improve the quality of the figures. Please see Figure 1 to figure 4 in the revised manuscript.

9. Minor comment 1: Why are sections 2.2 and 2.8 separate?

Response: Section 2.2 belongs to “Materials”, and Section 2.8 belongs to “Methods”. Please see the revised manuscript.

10. Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

Response: We agree to the reviewer’s suggestion. The caption has been added to Figure 2. In Figure 2, the horizontal axis represents the number of Ct values (cycles) and the vertical axis represents the number of template (copies), and their quantities are indicated in the figures. Please see Figure 2 in the revised manuscript.

Minor comments 3 (textual/narrative):

11. Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 13 in the revised manuscript.

12. Line 14: ..The established quadruplex RT-qPCR 'assay'...

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 15 in the revised manuscript.

13. Line 15: Use "reproducibility" instead of "repeatability"

Response: We agree to the reviewer’s suggestion. The "repeatability" has been replaced by "reproducibility" in the revised manuscript. Please see Lines 15, 192, 193, 281, 284, 287, 288, and 357 in the revised manuscript.

14. Line 40, 48, 57, 62: ..genome 'size'..

Response: We agree to the reviewer’s suggestion. The “size” has been added in the revised manuscript. Please see Lines 40, 49, 58, and 63 in the revised manuscript.

15. Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Response:  We agree to the reviewer’s suggestion. The "uneasy contamination/pollution" has been revised in the revised manuscript. Please see Lines 79, and 342 in the revised manuscript.

16. Line 85: ...and also obtains 'provides'.

Response: We agree to the reviewer’s suggestion. The “provides” has been added in the revised manuscript. Please see Lines 87 in the revised manuscript.

17. Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Response: We agree to the reviewer’s suggestion. The target genes were relatively conserved. The references have been cited in the revised manuscript. Please see Lines 120 in the revised manuscript.

18. Line 283-285: Cats pets are important companion.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 316 in the revised manuscript.

19. Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 322-323 in the revised manuscript.

20. Line 337: (make a new paragraph) In addition, co-infections.

Response: We agree to the reviewer’s suggestion. A new paragraph has been added in the revised manuscript. Please see Lines 403 in the revised manuscript.

21. Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.

Response: We agree to the reviewer’s suggestion. According to the reviewer’s suggestion (see the following suggestion), this sentence has been deleted. Please see Lines 443 in the revised manuscript.

22. Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Response: We agree to the reviewer’s suggestion. The sentences have been deleted in the revised manuscript. Please see Lines 441-445 in the revised manuscript.

23. Line 383-384: Uneven font sizes.

Response: We agree to the reviewer’s suggestion. The font size of this sentence has been corrected. Please see Lines 458-460 in the revised manuscript.

Comments on the Quality of English Language

24. Proof-reading is recommended.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of English language. Please see the revised manuscript.

 

Reviewer #3

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR.

1. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions.

Response: The purpose of RT-qPCR is to detect nucleic acids (RNA or DNA). Each technology has its function, and advantages, and also has its limitations. The RT-qPCR can detect viruses at RNA/DNA level, but not at protein level. To detect viruses at protein level, we need other technology and methods. This is beyond the scope of this study.

2. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

Response: The RT-qPCR can detect the target viruses at RNA/DNA level according to the principle of this technique. The purpose of this study is to develop a quadruplex RT-qPCR to detect FeKoV, FeAstV, FeBuV, and FRV. In this study, we did not detect viruses at protein level using other technology such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

3. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Response: In this study, we confirmed that FeBuV existed in Guangxi province using the developed quadruplex RT-qPCR to detect FeBuV in clinical samples. Now, we are trying to isolate and identify this virus in our laboratory.

 

Reviewer #4

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

Response: We agree to the reviewer’s suggestion. The prominent advantage of the developed RT-qPCR has been discussed in the Discussion part. Please see Lines 342-345 and 368-371 in the revised manuscript.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

Response: We agree to the reviewer’s suggestion. The clinical samples from Guangxi province in southern China were used to validate the application of the developed quadruplex RT-qPCR. This does not mean that the primers and probes have geographic limitation. The specific primers and probes were designed based on the genome sequences of FeKoV, FeAstV, FeBuV, and FRV which came from different countries (Supplementary Table S1-S4). The multiple sequence alignment was performed, and the conserved regions were selected to design specific primers and probes. They are suitable for the detection of the strains from different countries around the world. This has been discussed in the Discussion part. Please see Lines 371-380 in the revised manuscript.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

Response: We agree to the reviewer’s suggestion. The meaning of the developed assay has been discussed in the Discussion part. Please see Lines 381-385 in the revised manuscript.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

Response: We agree to the reviewer’s suggestion. The public health significance of the findings of these viruses has been discussed in the Discussion part in the revised part. Please see Lines 433-440 in the revised manuscript.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Response: We agree to the reviewer’s suggestion. The criteria for sample selection have been added in 2.2 Clinical Samples of the revised manuscript. Please see Lines 106-110 in the revised manuscript.

Minor Concerns:

6. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

Response: We agree to the reviewer’s suggestion. The definitions of "probit regression" and "coefficient variation" have been described in the revised manuscript. Please see Lines 188-191 and 197-200  in the revised manuscript.

7. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

Response: We agree to the reviewer’s suggestion. The tables have been checked, and used uniform formatting. The formatting will be corrected by the editor. Please see the revised manuscript.

8. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

Response: We agree to the reviewer’s suggestion. The references have been revised carefully, and the recent references have been updated. Please see the references in the revised manuscript.

9. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

Response: We agree to the reviewer’s suggestion. The section of the design of primers and probes has been revised. Please see Lines 112-125 in the revised manuscript.

10. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

Response: We agree to the reviewer’s suggestion. The workflow of the RT-qPCR development has been summarized and discussed in the Discussion part. Please see Lines 350-354 in the revised manuscript.

 Comments on the Quality of English Language

11. The language should be improved.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of the manuscript. Please see the revised manuscript.

 

Reviewer #5

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1869 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1. Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

Response: We agree to the reviewer’s suggestion. The four viruses can infect cats of all ages, but no vaccine is available for them now. This information has been added in the Introduction part of the revised manuscript. Please see Lines 71-75 in the revised manuscript.

2. What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

Response: We agree to the reviewer’s suggestion. All the samples were collected from subclinical or diseased cats. The feces, and anal/nasal swabs were collected from each cat, and mixed together for detection of FeKoV, FeAstV, FeBuV, and FRV. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869) and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diarrhea animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738) and 1.08% (8/738), respectively. The positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131) and 0.53% (6/1131), respectively. Please see Lines 291-296 in the revised manuscript.

3. Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]”. Why virus was associated with virus gene here?

Response: We agree to the reviewer’s suggestion. This sentence has been deleted. Please see Lines 58-59 in the revised manuscript.

4. “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative.

Response: We agree to the reviewer’s suggestion. “Vaccine Strains” has been changed to “Reference strains”. Please see Line 95 in the revised manuscript.

5. Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

Response: The amplification efficiency of real-time PCR is usually between 90%-110%, and the amplification efficiency of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were 109.798%, 109.965%, 108.551%, and 107.160%, respectively. They are normal values. Please see Lines 243-244 in the revised manuscript.

6. The font size of the last sentence of the main text looks smaller than the others.

Response: Sure. The font size has been corrected. Please see Lines 458-460 in the revised manuscript.

7. In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Response: We agree to the reviewer’s suggestion. “Date” has been substituted with “Year”. The Host column in the tables has been deleted. Please see the Supplementary Table S1-S4 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot , immunofluorescent staining cells in vitro, and flow cytometry. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Author Response

The Cover Letter

September 30, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the suggestions of the editor and the reviewers. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

  Comments:

1. Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 Response: According to the results of our test, the designed primers and probes could specifically detect the target genes. The fluorescence signals could obtain only from the four viruses and generate amplification curves, and no fluorescence signal could detect from other control viruses and negative controls. No cross-reactivity among the 4 targets within assays was observed, and no bleed-through fluorescence signal from neighboring channels occurred.

2. Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

Response: We agree to the reviewer’s suggestion. The genome sequences of FeKoV, FeAstV, FeBuV, and FRV were downloaded from the NCBI GenBank, and the multiple sequence alignment was performed. The conserved regions were selected to design specific primers and probes. Figure 1 shows the nucleotide sequence alignments of the partial FeKoV VP1 gene, FeAstV ORF2 gene, FeBuV VP2 gene, and FRV NSP4 gene, and the locations of the specific primers and probes designed for the quadruplex RT-qPCR. Please see Figure 1 in the revised manuscript.

In addition, the sequences of the designed primers and probes were performed BLAST analysis in the NCBI GenBank to confirm their specificity. Please see Lines 120-122 in the revised manuscript.

3. It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

Response: Besides FeKoV, FeAstV, FeBuV, and FRV, other viruses such as feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), and feline leukemia virus (FeLV) can also cause gastroenteritis showing clinical signs of vomiting, diarrhea, and dehydration. Their accurate diagnosis depends on the detection of pathogens in laboratory. In our previous report, a triplex quadruplex RT-qPCR was developed for the detection and differentiation of FPV, FCoV, and FeLV.  Therefore, the purpose of this study was to develop a quadruplex RT-qPCR for the detection and differentiation of FeKoV, FeAstV, FeBuV, and FRV. Please see Lines 334-337 in the revised manuscript.

The reference is as follows:

He M, Feng S, Shi K, Shi Y, Long F, Yin Y, Li Z. One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus. Vet World. 2024, 17(5):946-955.

4. Line 288-289: it would be helpful if the authors would define “PRV”.

Response: This is a spelling error. “PRV” should be “FRV”. It has been corrected. Please Lines 321 and 322 in the revised manuscript.

 

Reviewer #2

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 Major concerns

1. Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay.

Response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluated the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validated the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay.

 FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

2. Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions?

Response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

3. Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance.

Response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

4. Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

Major comments

5. Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity.

Response: In Figure 3, only one concentration of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV was used as positive controls, so the standard curves could not generate and show. In addition, no melting curve is generated in RT-qPCR/qPCR using specific TaqMan probe, so we cannot provide it here.

6. Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent.

Response: We agree to the reviewer’s suggestion. The sequences used for comparison to design the specific primers and probes were showed in figure 1, and the information of the virus strains including collected year were showed in Supplementary Table S1-S4. The accessed date of these sequences has been added in the revised manuscript. Please see Figure 1, Supplementary Table S1-S4, and Lines 112-123 in the revised manuscript.

7. Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation.

Response: We agree to the reviewer’s suggestion. The test results of 1869 clinical samples were showed in Table 5. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diseased animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738), and 1.08% (8/738), respectively, while the positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131), and 0.53% (6/1131), respectively. The positivity rates in diseased animals were higher than those in subclinical animals. Please see Lines 290-296 in the revised manuscript.

Minor comments

8. Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality.

Response: We agree to the reviewer’s suggestion. The Times New Roman was used in all figures, and the font sizes were different according to the size of the original images. In addition, Figure 2 and Figure 3 have been revised to improve the quality of the figures. Please see Figure 1 to figure 4 in the revised manuscript.

9. Minor comment 1: Why are sections 2.2 and 2.8 separate?

Response: Section 2.2 belongs to “Materials”, and Section 2.8 belongs to “Methods”. Please see the revised manuscript.

10. Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

Response: We agree to the reviewer’s suggestion. The caption has been added to Figure 2. In Figure 2, the horizontal axis represents the number of Ct values (cycles) and the vertical axis represents the number of template (copies), and their quantities are indicated in the figures. Please see Figure 2 in the revised manuscript.

Minor comments 3 (textual/narrative):

11. Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 13 in the revised manuscript.

12. Line 14: ..The established quadruplex RT-qPCR 'assay'...

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 15 in the revised manuscript.

13. Line 15: Use "reproducibility" instead of "repeatability"

Response: We agree to the reviewer’s suggestion. The "repeatability" has been replaced by "reproducibility" in the revised manuscript. Please see Lines 15, 192, 193, 281, 284, 287, 288, and 357 in the revised manuscript.

14. Line 40, 48, 57, 62: ..genome 'size'..

Response: We agree to the reviewer’s suggestion. The “size” has been added in the revised manuscript. Please see Lines 40, 49, 58, and 63 in the revised manuscript.

15. Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Response:  We agree to the reviewer’s suggestion. The "uneasy contamination/pollution" has been revised in the revised manuscript. Please see Lines 79, and 342 in the revised manuscript.

16. Line 85: ...and also obtains 'provides'.

Response: We agree to the reviewer’s suggestion. The “provides” has been added in the revised manuscript. Please see Lines 87 in the revised manuscript.

17. Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Response: We agree to the reviewer’s suggestion. The target genes were relatively conserved. The references have been cited in the revised manuscript. Please see Lines 120 in the revised manuscript.

18. Line 283-285: Cats pets are important companion.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 316 in the revised manuscript.

19. Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 322-323 in the revised manuscript.

20. Line 337: (make a new paragraph) In addition, co-infections.

Response: We agree to the reviewer’s suggestion. A new paragraph has been added in the revised manuscript. Please see Lines 403 in the revised manuscript.

21. Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.

Response: We agree to the reviewer’s suggestion. According to the reviewer’s suggestion (see the following suggestion), this sentence has been deleted. Please see Lines 443 in the revised manuscript.

22. Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Response: We agree to the reviewer’s suggestion. The sentences have been deleted in the revised manuscript. Please see Lines 441-445 in the revised manuscript.

23. Line 383-384: Uneven font sizes.

Response: We agree to the reviewer’s suggestion. The font size of this sentence has been corrected. Please see Lines 458-460 in the revised manuscript.

Comments on the Quality of English Language

24. Proof-reading is recommended.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of English language. Please see the revised manuscript.

 

Reviewer #3

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR.

1. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions.

Response: The purpose of RT-qPCR is to detect nucleic acids (RNA or DNA). Each technology has its function, and advantages, and also has its limitations. The RT-qPCR can detect viruses at RNA/DNA level, but not at protein level. To detect viruses at protein level, we need other technology and methods. This is beyond the scope of this study.

2. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

Response: The RT-qPCR can detect the target viruses at RNA/DNA level according to the principle of this technique. The purpose of this study is to develop a quadruplex RT-qPCR to detect FeKoV, FeAstV, FeBuV, and FRV. In this study, we did not detect viruses at protein level using other technology such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

3. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Response: In this study, we confirmed that FeBuV existed in Guangxi province using the developed quadruplex RT-qPCR to detect FeBuV in clinical samples. Now, we are trying to isolate and identify this virus in our laboratory.

 

Reviewer #4

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

Response: We agree to the reviewer’s suggestion. The prominent advantage of the developed RT-qPCR has been discussed in the Discussion part. Please see Lines 342-345 and 368-371 in the revised manuscript.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

Response: We agree to the reviewer’s suggestion. The clinical samples from Guangxi province in southern China were used to validate the application of the developed quadruplex RT-qPCR. This does not mean that the primers and probes have geographic limitation. The specific primers and probes were designed based on the genome sequences of FeKoV, FeAstV, FeBuV, and FRV which came from different countries (Supplementary Table S1-S4). The multiple sequence alignment was performed, and the conserved regions were selected to design specific primers and probes. They are suitable for the detection of the strains from different countries around the world. This has been discussed in the Discussion part. Please see Lines 371-380 in the revised manuscript.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

Response: We agree to the reviewer’s suggestion. The meaning of the developed assay has been discussed in the Discussion part. Please see Lines 381-385 in the revised manuscript.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

Response: We agree to the reviewer’s suggestion. The public health significance of the findings of these viruses has been discussed in the Discussion part in the revised part. Please see Lines 433-440 in the revised manuscript.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Response: We agree to the reviewer’s suggestion. The criteria for sample selection have been added in 2.2 Clinical Samples of the revised manuscript. Please see Lines 106-110 in the revised manuscript.

Minor Concerns:

6. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

Response: We agree to the reviewer’s suggestion. The definitions of "probit regression" and "coefficient variation" have been described in the revised manuscript. Please see Lines 188-191 and 197-200  in the revised manuscript.

7. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

Response: We agree to the reviewer’s suggestion. The tables have been checked, and used uniform formatting. The formatting will be corrected by the editor. Please see the revised manuscript.

8. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

Response: We agree to the reviewer’s suggestion. The references have been revised carefully, and the recent references have been updated. Please see the references in the revised manuscript.

9. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

Response: We agree to the reviewer’s suggestion. The section of the design of primers and probes has been revised. Please see Lines 112-125 in the revised manuscript.

10. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

Response: We agree to the reviewer’s suggestion. The workflow of the RT-qPCR development has been summarized and discussed in the Discussion part. Please see Lines 350-354 in the revised manuscript.

 Comments on the Quality of English Language

11. The language should be improved.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of the manuscript. Please see the revised manuscript.

 

Reviewer #5

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1869 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1. Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

Response: We agree to the reviewer’s suggestion. The four viruses can infect cats of all ages, but no vaccine is available for them now. This information has been added in the Introduction part of the revised manuscript. Please see Lines 71-75 in the revised manuscript.

2. What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

Response: We agree to the reviewer’s suggestion. All the samples were collected from subclinical or diseased cats. The feces, and anal/nasal swabs were collected from each cat, and mixed together for detection of FeKoV, FeAstV, FeBuV, and FRV. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869) and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diarrhea animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738) and 1.08% (8/738), respectively. The positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131) and 0.53% (6/1131), respectively. Please see Lines 291-296 in the revised manuscript.

3. Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]”. Why virus was associated with virus gene here?

Response: We agree to the reviewer’s suggestion. This sentence has been deleted. Please see Lines 58-59 in the revised manuscript.

4. “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative.

Response: We agree to the reviewer’s suggestion. “Vaccine Strains” has been changed to “Reference strains”. Please see Line 95 in the revised manuscript.

5. Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

Response: The amplification efficiency of real-time PCR is usually between 90%-110%, and the amplification efficiency of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were 109.798%, 109.965%, 108.551%, and 107.160%, respectively. They are normal values. Please see Lines 243-244 in the revised manuscript.

6. The font size of the last sentence of the main text looks smaller than the others.

Response: Sure. The font size has been corrected. Please see Lines 458-460 in the revised manuscript.

7. In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Response: We agree to the reviewer’s suggestion. “Date” has been substituted with “Year”. The Host column in the tables has been deleted. Please see the Supplementary Table S1-S4 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 

Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Minor Concerns:

1. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

2. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

3. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

4. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

5. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

 

Comments on the Quality of English Language

The language should be improved.

Author Response

The Cover Letter

September 30, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the suggestions of the editor and the reviewers. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

  Comments:

1. Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 Response: According to the results of our test, the designed primers and probes could specifically detect the target genes. The fluorescence signals could obtain only from the four viruses and generate amplification curves, and no fluorescence signal could detect from other control viruses and negative controls. No cross-reactivity among the 4 targets within assays was observed, and no bleed-through fluorescence signal from neighboring channels occurred.

2. Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

Response: We agree to the reviewer’s suggestion. The genome sequences of FeKoV, FeAstV, FeBuV, and FRV were downloaded from the NCBI GenBank, and the multiple sequence alignment was performed. The conserved regions were selected to design specific primers and probes. Figure 1 shows the nucleotide sequence alignments of the partial FeKoV VP1 gene, FeAstV ORF2 gene, FeBuV VP2 gene, and FRV NSP4 gene, and the locations of the specific primers and probes designed for the quadruplex RT-qPCR. Please see Figure 1 in the revised manuscript.

In addition, the sequences of the designed primers and probes were performed BLAST analysis in the NCBI GenBank to confirm their specificity. Please see Lines 120-122 in the revised manuscript.

3. It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

Response: Besides FeKoV, FeAstV, FeBuV, and FRV, other viruses such as feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), and feline leukemia virus (FeLV) can also cause gastroenteritis showing clinical signs of vomiting, diarrhea, and dehydration. Their accurate diagnosis depends on the detection of pathogens in laboratory. In our previous report, a triplex quadruplex RT-qPCR was developed for the detection and differentiation of FPV, FCoV, and FeLV.  Therefore, the purpose of this study was to develop a quadruplex RT-qPCR for the detection and differentiation of FeKoV, FeAstV, FeBuV, and FRV. Please see Lines 334-337 in the revised manuscript.

The reference is as follows:

He M, Feng S, Shi K, Shi Y, Long F, Yin Y, Li Z. One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus. Vet World. 2024, 17(5):946-955.

4. Line 288-289: it would be helpful if the authors would define “PRV”.

Response: This is a spelling error. “PRV” should be “FRV”. It has been corrected. Please Lines 321 and 322 in the revised manuscript.

 

Reviewer #2

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 Major concerns

1. Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay.

Response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluated the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validated the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay.

 FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

2. Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions?

Response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

3. Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance.

Response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

4. Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

Major comments

5. Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity.

Response: In Figure 3, only one concentration of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV was used as positive controls, so the standard curves could not generate and show. In addition, no melting curve is generated in RT-qPCR/qPCR using specific TaqMan probe, so we cannot provide it here.

6. Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent.

Response: We agree to the reviewer’s suggestion. The sequences used for comparison to design the specific primers and probes were showed in figure 1, and the information of the virus strains including collected year were showed in Supplementary Table S1-S4. The accessed date of these sequences has been added in the revised manuscript. Please see Figure 1, Supplementary Table S1-S4, and Lines 112-123 in the revised manuscript.

7. Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation.

Response: We agree to the reviewer’s suggestion. The test results of 1869 clinical samples were showed in Table 5. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diseased animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738), and 1.08% (8/738), respectively, while the positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131), and 0.53% (6/1131), respectively. The positivity rates in diseased animals were higher than those in subclinical animals. Please see Lines 290-296 in the revised manuscript.

Minor comments

8. Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality.

Response: We agree to the reviewer’s suggestion. The Times New Roman was used in all figures, and the font sizes were different according to the size of the original images. In addition, Figure 2 and Figure 3 have been revised to improve the quality of the figures. Please see Figure 1 to figure 4 in the revised manuscript.

9. Minor comment 1: Why are sections 2.2 and 2.8 separate?

Response: Section 2.2 belongs to “Materials”, and Section 2.8 belongs to “Methods”. Please see the revised manuscript.

10. Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

Response: We agree to the reviewer’s suggestion. The caption has been added to Figure 2. In Figure 2, the horizontal axis represents the number of Ct values (cycles) and the vertical axis represents the number of template (copies), and their quantities are indicated in the figures. Please see Figure 2 in the revised manuscript.

Minor comments 3 (textual/narrative):

11. Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 13 in the revised manuscript.

12. Line 14: ..The established quadruplex RT-qPCR 'assay'...

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 15 in the revised manuscript.

13. Line 15: Use "reproducibility" instead of "repeatability"

Response: We agree to the reviewer’s suggestion. The "repeatability" has been replaced by "reproducibility" in the revised manuscript. Please see Lines 15, 192, 193, 281, 284, 287, 288, and 357 in the revised manuscript.

14. Line 40, 48, 57, 62: ..genome 'size'..

Response: We agree to the reviewer’s suggestion. The “size” has been added in the revised manuscript. Please see Lines 40, 49, 58, and 63 in the revised manuscript.

15. Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Response:  We agree to the reviewer’s suggestion. The "uneasy contamination/pollution" has been revised in the revised manuscript. Please see Lines 79, and 342 in the revised manuscript.

16. Line 85: ...and also obtains 'provides'.

Response: We agree to the reviewer’s suggestion. The “provides” has been added in the revised manuscript. Please see Lines 87 in the revised manuscript.

17. Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Response: We agree to the reviewer’s suggestion. The target genes were relatively conserved. The references have been cited in the revised manuscript. Please see Lines 120 in the revised manuscript.

18. Line 283-285: Cats pets are important companion.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 316 in the revised manuscript.

19. Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 322-323 in the revised manuscript.

20. Line 337: (make a new paragraph) In addition, co-infections.

Response: We agree to the reviewer’s suggestion. A new paragraph has been added in the revised manuscript. Please see Lines 403 in the revised manuscript.

21. Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.

Response: We agree to the reviewer’s suggestion. According to the reviewer’s suggestion (see the following suggestion), this sentence has been deleted. Please see Lines 443 in the revised manuscript.

22. Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Response: We agree to the reviewer’s suggestion. The sentences have been deleted in the revised manuscript. Please see Lines 441-445 in the revised manuscript.

23. Line 383-384: Uneven font sizes.

Response: We agree to the reviewer’s suggestion. The font size of this sentence has been corrected. Please see Lines 458-460 in the revised manuscript.

Comments on the Quality of English Language

24. Proof-reading is recommended.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of English language. Please see the revised manuscript.

 

Reviewer #3

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR.

1. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions.

Response: The purpose of RT-qPCR is to detect nucleic acids (RNA or DNA). Each technology has its function, and advantages, and also has its limitations. The RT-qPCR can detect viruses at RNA/DNA level, but not at protein level. To detect viruses at protein level, we need other technology and methods. This is beyond the scope of this study.

2. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

Response: The RT-qPCR can detect the target viruses at RNA/DNA level according to the principle of this technique. The purpose of this study is to develop a quadruplex RT-qPCR to detect FeKoV, FeAstV, FeBuV, and FRV. In this study, we did not detect viruses at protein level using other technology such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

3. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Response: In this study, we confirmed that FeBuV existed in Guangxi province using the developed quadruplex RT-qPCR to detect FeBuV in clinical samples. Now, we are trying to isolate and identify this virus in our laboratory.

 

Reviewer #4

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

Response: We agree to the reviewer’s suggestion. The prominent advantage of the developed RT-qPCR has been discussed in the Discussion part. Please see Lines 342-345 and 368-371 in the revised manuscript.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

Response: We agree to the reviewer’s suggestion. The clinical samples from Guangxi province in southern China were used to validate the application of the developed quadruplex RT-qPCR. This does not mean that the primers and probes have geographic limitation. The specific primers and probes were designed based on the genome sequences of FeKoV, FeAstV, FeBuV, and FRV which came from different countries (Supplementary Table S1-S4). The multiple sequence alignment was performed, and the conserved regions were selected to design specific primers and probes. They are suitable for the detection of the strains from different countries around the world. This has been discussed in the Discussion part. Please see Lines 371-380 in the revised manuscript.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

Response: We agree to the reviewer’s suggestion. The meaning of the developed assay has been discussed in the Discussion part. Please see Lines 381-385 in the revised manuscript.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

Response: We agree to the reviewer’s suggestion. The public health significance of the findings of these viruses has been discussed in the Discussion part in the revised part. Please see Lines 433-440 in the revised manuscript.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Response: We agree to the reviewer’s suggestion. The criteria for sample selection have been added in 2.2 Clinical Samples of the revised manuscript. Please see Lines 106-110 in the revised manuscript.

Minor Concerns:

6. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

Response: We agree to the reviewer’s suggestion. The definitions of "probit regression" and "coefficient variation" have been described in the revised manuscript. Please see Lines 188-191 and 197-200  in the revised manuscript.

7. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

Response: We agree to the reviewer’s suggestion. The tables have been checked, and used uniform formatting. The formatting will be corrected by the editor. Please see the revised manuscript.

8. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

Response: We agree to the reviewer’s suggestion. The references have been revised carefully, and the recent references have been updated. Please see the references in the revised manuscript.

9. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

Response: We agree to the reviewer’s suggestion. The section of the design of primers and probes has been revised. Please see Lines 112-125 in the revised manuscript.

10. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

Response: We agree to the reviewer’s suggestion. The workflow of the RT-qPCR development has been summarized and discussed in the Discussion part. Please see Lines 350-354 in the revised manuscript.

 Comments on the Quality of English Language

11. The language should be improved.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of the manuscript. Please see the revised manuscript.

 

Reviewer #5

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1869 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1. Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

Response: We agree to the reviewer’s suggestion. The four viruses can infect cats of all ages, but no vaccine is available for them now. This information has been added in the Introduction part of the revised manuscript. Please see Lines 71-75 in the revised manuscript.

2. What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

Response: We agree to the reviewer’s suggestion. All the samples were collected from subclinical or diseased cats. The feces, and anal/nasal swabs were collected from each cat, and mixed together for detection of FeKoV, FeAstV, FeBuV, and FRV. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869) and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diarrhea animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738) and 1.08% (8/738), respectively. The positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131) and 0.53% (6/1131), respectively. Please see Lines 291-296 in the revised manuscript.

3. Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]”. Why virus was associated with virus gene here?

Response: We agree to the reviewer’s suggestion. This sentence has been deleted. Please see Lines 58-59 in the revised manuscript.

4. “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative.

Response: We agree to the reviewer’s suggestion. “Vaccine Strains” has been changed to “Reference strains”. Please see Line 95 in the revised manuscript.

5. Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

Response: The amplification efficiency of real-time PCR is usually between 90%-110%, and the amplification efficiency of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were 109.798%, 109.965%, 108.551%, and 107.160%, respectively. They are normal values. Please see Lines 243-244 in the revised manuscript.

6. The font size of the last sentence of the main text looks smaller than the others.

Response: Sure. The font size has been corrected. Please see Lines 458-460 in the revised manuscript.

7. In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Response: We agree to the reviewer’s suggestion. “Date” has been substituted with “Year”. The Host column in the tables has been deleted. Please see the Supplementary Table S1-S4 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1899 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1.       Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

2.       What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

3.       Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]. “Why virus was associated with virus gene here?

4.       “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative./span/p p class="MsoListParagraphCxSpMiddle" style="margin-left: 21.3pt; mso-add-space: auto; text-align: justify; text-justify: inter-ideograph; text-indent: -21.0pt; mso-list: l0 level1 lfo1"5.       Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

6.       The font size of the last sentence of the main text looks smaller than the others.

7.       In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Author Response

The Cover Letter

September 30, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the suggestions of the editor and the reviewers. The details are as follows.

 

Reviewer #1

Comments and Suggestions for Authors

In this manuscript, the authors established a multiplex RT-qPCR assay that enables clinical labs to quickly diagnose 4 enteric viral pathogens as well as co-infection among these viruses in cats. This new quadruplex assay increases test capacity, saves resources and reduces turnaround time compared with reported reference singleplex assays. It is a useful tool that would facilitate surveillance of enteric viruses in cats and potential cross-species transmission of these viruses to human. This article is well-written, but still has some questions that need to be addressed.

  Comments:

1. Figure 2 and 3: did the authors observe any cross-reactivity among the 4 targets within assays? Sometimes bleed-through fluorescence signals from neighboring channels would occur when multiple primer-probe sets are used simultaneously.

 Response: According to the results of our test, the designed primers and probes could specifically detect the target genes. The fluorescence signals could obtain only from the four viruses and generate amplification curves, and no fluorescence signal could detect from other control viruses and negative controls. No cross-reactivity among the 4 targets within assays was observed, and no bleed-through fluorescence signal from neighboring channels occurred.

2. Figure 3: the authors stated that this assay has high specificity as no cross-reactivity was observed in five feline-related viruses other than the 4 targeted viruses. To confirm its specificity, did the authors perform BLAST analysis on these primer sequences?

Response: We agree to the reviewer’s suggestion. The genome sequences of FeKoV, FeAstV, FeBuV, and FRV were downloaded from the NCBI GenBank, and the multiple sequence alignment was performed. The conserved regions were selected to design specific primers and probes. Figure 1 shows the nucleotide sequence alignments of the partial FeKoV VP1 gene, FeAstV ORF2 gene, FeBuV VP2 gene, and FRV NSP4 gene, and the locations of the specific primers and probes designed for the quadruplex RT-qPCR. Please see Figure 1 in the revised manuscript.

In addition, the sequences of the designed primers and probes were performed BLAST analysis in the NCBI GenBank to confirm their specificity. Please see Lines 120-122 in the revised manuscript.

3. It would be helpful if the authors could elaborate in the discussion or introduction on the reasons that they chose these four viruses over other common viral causes of gastroenteritis in cats, e.g., feline panleukopenia virus, feline enteric coronavirus and feline leukemia virus.

Response: Besides FeKoV, FeAstV, FeBuV, and FRV, other viruses such as feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), and feline leukemia virus (FeLV) can also cause gastroenteritis showing clinical signs of vomiting, diarrhea, and dehydration. Their accurate diagnosis depends on the detection of pathogens in laboratory. In our previous report, a triplex quadruplex RT-qPCR was developed for the detection and differentiation of FPV, FCoV, and FeLV.  Therefore, the purpose of this study was to develop a quadruplex RT-qPCR for the detection and differentiation of FeKoV, FeAstV, FeBuV, and FRV. Please see Lines 334-337 in the revised manuscript.

The reference is as follows:

He M, Feng S, Shi K, Shi Y, Long F, Yin Y, Li Z. One-step triplex TaqMan quantitative reverse transcription polymerase chain reaction for the detection of feline coronavirus, feline panleukopenia virus, and feline leukemia virus. Vet World. 2024, 17(5):946-955.

4. Line 288-289: it would be helpful if the authors would define “PRV”.

Response: This is a spelling error. “PRV” should be “FRV”. It has been corrected. Please Lines 321 and 322 in the revised manuscript.

 

Reviewer #2

Comments and Suggestions for Authors

The study by Shi et al. describes the development of a multiplex RT-qPCR assay to detect four natural viruses endemic to domesticated felines: Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV). The described assay will be useful in its application and meets a well-argued need. The study is competently written in parts, but the materials and methods section lacks detail. The figures presented are uneven in quality and presentation. The authors present compelling evidence that the assay is able to detect these viruses from clinical samples with precision and sensitivity, but several issues were identified in the methodology that should be addressed. Please refer to the comments below.

 Major concerns

1. Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay.

Response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluated the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validated the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay.

 FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

2. Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions?

Response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

3. Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance.

Response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

4. Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included.

Response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

Major comments

5. Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity.

Response: In Figure 3, only one concentration of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV was used as positive controls, so the standard curves could not generate and show. In addition, no melting curve is generated in RT-qPCR/qPCR using specific TaqMan probe, so we cannot provide it here.

6. Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent.

Response: We agree to the reviewer’s suggestion. The sequences used for comparison to design the specific primers and probes were showed in figure 1, and the information of the virus strains including collected year were showed in Supplementary Table S1-S4. The accessed date of these sequences has been added in the revised manuscript. Please see Figure 1, Supplementary Table S1-S4, and Lines 112-123 in the revised manuscript.

7. Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation.

Response: We agree to the reviewer’s suggestion. The test results of 1869 clinical samples were showed in Table 5. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diseased animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738), and 1.08% (8/738), respectively, while the positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131), and 0.53% (6/1131), respectively. The positivity rates in diseased animals were higher than those in subclinical animals. Please see Lines 290-296 in the revised manuscript.

Minor comments

8. Figures: Use font styles and sizes consistently (preferably the one used in Figure 4). Figures 2-3 appear to be low-quality.

Response: We agree to the reviewer’s suggestion. The Times New Roman was used in all figures, and the font sizes were different according to the size of the original images. In addition, Figure 2 and Figure 3 have been revised to improve the quality of the figures. Please see Figure 1 to figure 4 in the revised manuscript.

9. Minor comment 1: Why are sections 2.2 and 2.8 separate?

Response: Section 2.2 belongs to “Materials”, and Section 2.8 belongs to “Methods”. Please see the revised manuscript.

10. Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

Response: We agree to the reviewer’s suggestion. The caption has been added to Figure 2. In Figure 2, the horizontal axis represents the number of Ct values (cycles) and the vertical axis represents the number of template (copies), and their quantities are indicated in the figures. Please see Figure 2 in the revised manuscript.

Minor comments 3 (textual/narrative):

11. Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 13 in the revised manuscript.

12. Line 14: ..The established quadruplex RT-qPCR 'assay'...

Response: We agree to the reviewer’s suggestion. The “assay” has been added. Please see Line 15 in the revised manuscript.

13. Line 15: Use "reproducibility" instead of "repeatability"

Response: We agree to the reviewer’s suggestion. The "repeatability" has been replaced by "reproducibility" in the revised manuscript. Please see Lines 15, 192, 193, 281, 284, 287, 288, and 357 in the revised manuscript.

14. Line 40, 48, 57, 62: ..genome 'size'..

Response: We agree to the reviewer’s suggestion. The “size” has been added in the revised manuscript. Please see Lines 40, 49, 58, and 63 in the revised manuscript.

15. Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Response:  We agree to the reviewer’s suggestion. The "uneasy contamination/pollution" has been revised in the revised manuscript. Please see Lines 79, and 342 in the revised manuscript.

16. Line 85: ...and also obtains 'provides'.

Response: We agree to the reviewer’s suggestion. The “provides” has been added in the revised manuscript. Please see Lines 87 in the revised manuscript.

17. Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Response: We agree to the reviewer’s suggestion. The target genes were relatively conserved. The references have been cited in the revised manuscript. Please see Lines 120 in the revised manuscript.

18. Line 283-285: Cats pets are important companion.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 316 in the revised manuscript.

19. Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Response: We agree to the reviewer’s suggestion. This sentence has been revised in the revised manuscript. Please see Lines 322-323 in the revised manuscript.

20. Line 337: (make a new paragraph) In addition, co-infections.

Response: We agree to the reviewer’s suggestion. A new paragraph has been added in the revised manuscript. Please see Lines 403 in the revised manuscript.

21. Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.

Response: We agree to the reviewer’s suggestion. According to the reviewer’s suggestion (see the following suggestion), this sentence has been deleted. Please see Lines 443 in the revised manuscript.

22. Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Response: We agree to the reviewer’s suggestion. The sentences have been deleted in the revised manuscript. Please see Lines 441-445 in the revised manuscript.

23. Line 383-384: Uneven font sizes.

Response: We agree to the reviewer’s suggestion. The font size of this sentence has been corrected. Please see Lines 458-460 in the revised manuscript.

Comments on the Quality of English Language

24. Proof-reading is recommended.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of English language. Please see the revised manuscript.

 

Reviewer #3

Comments and Suggestions for Authors

It is very important that the authors’ observations of the emerging problems that come from the increasing number of cats. It is very cool that the authors have developed a quadruplex RT-qPCR.

1. But the RT-qPCR has its limitations since this tech investigates the problem from RNA level instead of protein level which is much closer to the diagnose of the diseased conditions.

Response: The purpose of RT-qPCR is to detect nucleic acids (RNA or DNA). Each technology has its function, and advantages, and also has its limitations. The RT-qPCR can detect viruses at RNA/DNA level, but not at protein level. To detect viruses at protein level, we need other technology and methods. This is beyond the scope of this study.

2. In addition, if only taking a look at the RNA level, it definitely ignores the post-transcriptional modifications. I am wondering if the authors can add some protein level experiments and results such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

Response: The RT-qPCR can detect the target viruses at RNA/DNA level according to the principle of this technique. The purpose of this study is to develop a quadruplex RT-qPCR to detect FeKoV, FeAstV, FeBuV, and FRV. In this study, we did not detect viruses at protein level using other technology such as immunoblot, immunofluorescent staining cells in vitro, and flow cytometry.

3. Since the author at the end mentioned that “this is the first report that FeBuV exists in Guangxi province, southern China.” Can the authors start to test FeBuV using the protein level experiment to see if it will be working?

Response: In this study, we confirmed that FeBuV existed in Guangxi province using the developed quadruplex RT-qPCR to detect FeBuV in clinical samples. Now, we are trying to isolate and identify this virus in our laboratory.

 

Reviewer #4

Comments and Suggestions for Authors

The manuscript (microbiolres-3207179) titled "Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV" describes the development and validation of a real-time quantitative RT-PCR assay for the simultaneous detection of four feline viruses: Feline kobuvirus (FeKoV), Feline astrovirus (FeAstV), Feline bufavirus (FeBuV), and Feline rotavirus (FRV). These viruses are responsible for causing gastroenteritis in cats, leading to symptoms like vomiting, diarrhoea, and dehydration. The study aims to provide an efficient detection method, demonstrating that the assay is highly specific, sensitive, and reliable. It was applied to 1869 clinical samples, and the results were compared to existing reference assays, showing high coincidence rates.

 Major Concerns:

1. Multiplex RT-qPCR is a commonly used method in virology, and the manuscript does not clearly differentiate this assay from existing methods, especially since similar detection methods for other feline viruses are well-established. More discussion on the novelty of combining these specific viruses for simultaneous detection is required.

Response: We agree to the reviewer’s suggestion. The prominent advantage of the developed RT-qPCR has been discussed in the Discussion part. Please see Lines 342-345 and 368-371 in the revised manuscript.

2. The study is focused on samples from Guangxi province, China. This may limit its applicability to broader geographic regions or global contexts, and there is a lack of discussion on how these findings could be generalized beyond this region.

Response: We agree to the reviewer’s suggestion. The clinical samples from Guangxi province in southern China were used to validate the application of the developed quadruplex RT-qPCR. This does not mean that the primers and probes have geographic limitation. The specific primers and probes were designed based on the genome sequences of FeKoV, FeAstV, FeBuV, and FRV which came from different countries (Supplementary Table S1-S4). The multiple sequence alignment was performed, and the conserved regions were selected to design specific primers and probes. They are suitable for the detection of the strains from different countries around the world. This has been discussed in the Discussion part. Please see Lines 371-380 in the revised manuscript.

3. While the study proves the technical efficacy of the assay, it lacks a deeper exploration of the clinical implications, particularly regarding the prevalence of these viruses and co-infections. A more comprehensive analysis of how this detection method will impact the diagnosis and treatment of gastroenteritis in cats is needed.

Response: We agree to the reviewer’s suggestion. The meaning of the developed assay has been discussed in the Discussion part. Please see Lines 381-385 in the revised manuscript.

4. The study briefly mentions the zoonotic potential of some of the viruses, but does not delve into the public health significance of these findings. This should be more thoroughly discussed, especially with the increasing prevalence of companion animals in human households.

Response: We agree to the reviewer’s suggestion. The public health significance of the findings of these viruses has been discussed in the Discussion part in the revised part. Please see Lines 433-440 in the revised manuscript.

5. The manuscript does not elaborate on the criteria for sample selection. A more detailed description of the sample population (age, breed, clinical symptoms, etc.) would strengthen the clinical relevance of the results.

Response: We agree to the reviewer’s suggestion. The criteria for sample selection have been added in 2.2 Clinical Samples of the revised manuscript. Please see Lines 106-110 in the revised manuscript.

Minor Concerns:

6. Certain technical terms such as "probit regression" and "coefficient variation" are mentioned without sufficient explanation. Including clearer definitions or context for readers unfamiliar with these terms would improve accessibility.

Response: We agree to the reviewer’s suggestion. The definitions of "probit regression" and "coefficient variation" have been described in the revised manuscript. Please see Lines 188-191 and 197-200  in the revised manuscript.

7. The manuscript contains inconsistencies in the formatting of the tables, such as Table 4 (repeatability analysis), which may confuse the reader. Uniform formatting is needed for clarity.

Response: We agree to the reviewer’s suggestion. The tables have been checked, and used uniform formatting. The formatting will be corrected by the editor. Please see the revised manuscript.

8. Some references are outdated or incomplete, which could undermine the reliability of the literature review. The reference list should be updated to include more recent and relevant studies.

Response: We agree to the reviewer’s suggestion. The references have been revised carefully, and the recent references have been updated. Please see the references in the revised manuscript.

9. Some of the procedural details, such as the design of primers and probes, are overly detailed and could be condensed. Streamlining this section would improve readability.

Response: We agree to the reviewer’s suggestion. The section of the design of primers and probes has been revised. Please see Lines 112-125 in the revised manuscript.

10. It would be better to provide a graphical abstract or flowchart summarizing the workflow of the RT-qPCR development, and testing would greatly enhance comprehension for visual learners.

Response: We agree to the reviewer’s suggestion. The workflow of the RT-qPCR development has been summarized and discussed in the Discussion part. Please see Lines 350-354 in the revised manuscript.

 Comments on the Quality of English Language

11. The language should be improved.

Response: We agree to the reviewer’s suggestion. The manuscript has been revised carefully to improve the quality of the manuscript. Please see the revised manuscript.

 

Reviewer #5

Comments and Suggestions for Authors

In the ms entitled “Development of a Quadruplex RT-qPCR for the Detection of FeKoV, FeAstV, FeBuV, and FRV”, Shi et. al. established a quadruplex RT-qPCR method for the simultaneous detection of four feline enteroviruses and applied it in the detection of 1869 clinical samples, showing good coincidence rates with that of the reference assay. The method established provides a new detection mean for feline enteritis associated with FeKoV, FeAstV, FeBuV, and FRV. The ms was well-written and the results were well presented.

Some comments are list below:

1. Currently, how harmful are the four viruses to cats, especially with different ages? Are there vaccines form them? Please introduce them in the introduction section.

Response: We agree to the reviewer’s suggestion. The four viruses can infect cats of all ages, but no vaccine is available for them now. This information has been added in the Introduction part of the revised manuscript. Please see Lines 71-75 in the revised manuscript.

2. What are the positive rates of the four viruses, FeKoV, FeAstV, FeBuV, and FRV, in diarrhea samples used in this study? And what about the health animals?

Response: We agree to the reviewer’s suggestion. All the samples were collected from subclinical or diseased cats. The feces, and anal/nasal swabs were collected from each cat, and mixed together for detection of FeKoV, FeAstV, FeBuV, and FRV. The positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869) and 0.75% (14/1869), respectively. Of these positive samples, the positive rates of FeKoV, FeAstV, FeBuV and FRV in the samples from diarrhea animals were 3.52% (26/738), 14.77% (109/738), 0.27% (2/738) and 1.08% (8/738), respectively. The positive rates in the samples from subclinical animals were 0.88% (10/1131), 5.84% (66/1131), 0.35% (4/1131) and 0.53% (6/1131), respectively. Please see Lines 291-296 in the revised manuscript.

3. Line 57-58, “FeBuV is a new parvovirus, which is associated with canine bufavirus gene [24, 25]”. Why virus was associated with virus gene here?

Response: We agree to the reviewer’s suggestion. This sentence has been deleted. Please see Lines 58-59 in the revised manuscript.

4. “2.1 Vaccine Strains” is not accurate, as positive clinical sample also included in this section. Reference strains maybe as alternative.

Response: We agree to the reviewer’s suggestion. “Vaccine Strains” has been changed to “Reference strains”. Please see Line 95 in the revised manuscript.

5. Line 220-221, the amplification efficiency of all viruses are greater than 100%, which might due to errors in sample dilution or non-specific product amplification, such primer dimer production. Please discuss.

Response: The amplification efficiency of real-time PCR is usually between 90%-110%, and the amplification efficiency of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were 109.798%, 109.965%, 108.551%, and 107.160%, respectively. They are normal values. Please see Lines 243-244 in the revised manuscript.

6. The font size of the last sentence of the main text looks smaller than the others.

Response: Sure. The font size has been corrected. Please see Lines 458-460 in the revised manuscript.

7. In the headline of Supplementary Tables, substitute Date with Year. In addition, it is preferred to delete the Host column as they are all feline except one environment which cannot be categorized into host feline.

Response: We agree to the reviewer’s suggestion. “Date” has been substituted with “Year”. The Host column in the tables has been deleted. Please see the Supplementary Table S1-S4 in the revised manuscript.

 

Best regards,

 

Kaichuang Shi

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript is improved in quality due to sincere efforts from the authors. The discussion section is significantly better. A new comment to this revised version and responses to the author’s responses are included below.

 

Major concern (version 2)

 

One gold-standard to demonstrate PCR assay sensitivity, is to ensure whether the primers are amplifying the correct target. Can the authors sequence the amplicons that were generated from clinical samples, perform multiple sequence alignment and BLAST analysis to confirm the specificity of the primers? This would make the assay specificity argument more compelling. 

 

Major concerns (version 1)

 

Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay. FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples. 

 

Author’s response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluate the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validate the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay. We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

 

The fact that the authors use a One-step RT PCR setup does not exclude the variability introduced in sample purification and RT-steps. Can the authors confirm whether the assay sensitivity is comparable between the plasmid vs RNA templates? 

 

Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions? 

 

Author’s response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

 

The author’s response and revision in the text does not address the original question. The specificity can only really be tested by using varying concentrations of target (FeKoV, FeAstV, FeBuV, and FRV) and non-target/contaminating (FPV, FCoV, FBoV, FCV, and FHV) template. In the absence of such data, it is unclear whether or not this assay can detect FeKoV, FeAstV, FeBuV, and FRV in clinical samples that have varying loads of FPV, FCoV, FBoV, FCV, and FHV.

 

Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance. 

 

Author’s response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

 

The authors should include this statement in their materials and methods and refer to the results from all combinations in the supplemental data. The sensitivity at lower genome copies in the context of single vs co-infections may vary.

 

Major concern 4: The clinical samples section lacks detail. Since this is the first and only instance that the authors perform the assay using RNA, a detailed RNA extraction and RT assay protocol needs to be included. 

 

Author’s response: We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR assay was a one-step RT-qPCR, and the extracted RNA was used as template to test the target fragment. The reversed transcription (42 °C for 5 min; 95 °C for 10 s) and amplification (40 cycles of 95 °C for 5 s, 56 °C for 30 s) were preformed continuously in one tube. This has been added in the revised manuscript. Please see Lines 203-208 in the revised manuscript.

 

This has been resolved to satisfaction.

 

Major comments

 

Major comment 1: Figure 3. The authors need to show the standard curves for p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV. Without it, it is difficult to gauge the dynamic range for this assay. Please also include the melt curves for each target-primer pair (supplemental data is fine). This is required to gauge specificity. 

 

Resolved to satisfaction. 

 

Major comment 2: Line 106: Please specify how many sequences and the collection period. Are these viral sequences from recent outbreaks? Also in Figure 1, please consider representing the aligned sequences adjacent to a phylogenetic tree (one for each virus species). The current presentation provides no sense about phylogenetic relationships between strains used to design the reagent. 

 

Resolved to satisfaction. 

 

Major comment 3: What is the breakdown of the detection results/positivity rate based on disease state of the animals tested? Presumably the authors have this data from the time of sample collection. Does it correlate well with the RT-qPCR assay results? This data will be useful in terms of real-world validation. 

 

Resolved to satisfaction. 

 

Minor comments

 

Minor comment 1: Why are sections 2.2 and 2.8 separate? 

 

Resolved to satisfaction. 

 

Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

 

The figure caption for panels A-D shows Cycle (Ct) values on the horizontal-axis, while panel E shows Cycle (Ct) values on the vertical-axis. Please make this distinction in the legend. Furthermore, increase the panel sizes for A-D to match E. The font sizes and figure labels are oddly placed inside the plot. Please place it in the top upper left corner outside of each plot.

 

Minor comments 3 (textual/narrative): 

 

Line 13: ..quantitative RT-PCR (RT-qPCR) 'assay' was developed..

Line 14: ..The established quadruplex RT-qPCR 'assay'...

Line 15: Use "reproducibility" instead of "repeatability"

Line 40, 48, 57, 62: ..genome 'size'..

Line 78-Line 303: What do the authors mean by "uneasy contamination/pollution" Please define or use different verbiage

Line 85: ...and also obtains 'provides'..

Line 112: Provide rationale as to why these genes were chosen – are they more conserved and less likely to change, recombine or undergo reassortment? Provide citations.

Line 283-285: Cats pets are important companion..

Lines 288-289: and the infection of PRV in cat pets might also threaten human's health, and cause problem of common health security.

Line 337: (make a new paragraph) In addition, co-infections..

Line 369: “Furthermore, FRV was confirmed to be zoonotic potential.” – are the authors referring to this study or a different one? If it is the latter, please cite. If it is the former, the supporting data is missing since I do not see any samples that were obtained from human or other animal species.  

Lines 367-371: Eliminate – too repetitive (see comment above for statement made about line 369)

Line 383-384: Uneven font sizes.

 

These have been addressed to satisfaction.

Comments on the Quality of English Language

None

Author Response

The Cover Letter

October 12, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the reviewers’ suggestions. The details are follows.

 

Reviewer #2 (Round 2)

 

Comments and Suggestions for Authors

The revised manuscript is improved in quality due to sincere efforts from the authors. The discussion section is significantly better. A new comment to this revised version and responses to the author’s responses are included below.

 

Major concern (version 2)

 

One gold-standard to demonstrate PCR assay sensitivity, is to ensure whether the primers are amplifying the correct target. Can the authors sequence the amplicons that were generated from clinical samples, perform multiple sequence alignment and BLAST analysis to confirm the specificity of the primers? This would make the assay specificity argument more compelling. 

Response: We agree to the reviewer’s suggestion. This job has been done during our experiments. In this study, a total of 1869 clinical samples were tested using the developed quadruplex RT-qPCR, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, we selected some positive samples to amplify and sequence the target fragments, and performed multiple sequence alignment and BLAST analysis to confirm that the amplified fragments were correct. This content has been added in the revised manuscript. Please see Lines 321-325 in the revised manuscript.

 

Major concerns (version 1)

 

Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay. FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples. 

 

Author’s response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluate the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validate the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay. We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

 

The fact that the authors use a One-step RT PCR setup does not exclude the variability introduced in sample purification and RT-steps. Can the authors confirm whether the assay sensitivity is comparable between the plasmid vs RNA templates? 

Response: In this study, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluate the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. We are sorry that we did not use RNA to analyze the assay’s sensitivity, so we could not compare the results between plasmids and RNA templates in this study.

 

Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions? 

 

Author’s response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

 

The author’s response and revision in the text does not address the original question. The specificity can only really be tested by using varying concentrations of target (FeKoV, FeAstV, FeBuV, and FRV) and non-target/contaminating (FPV, FCoV, FBoV, FCV, and FHV) template. In the absence of such data, it is unclear whether or not this assay can detect FeKoV, FeAstV, FeBuV, and FRV in clinical samples that have varying loads of FPV, FCoV, FBoV, FCV, and FHV.

Response: We agree to the reviewer’s suggestion. The vaccine solutions of FPV, FHV, and FCV, and the positive clinical samples of FCoV, and FBoV were used to extracted the total nucleic acids (DNA/RNA), and used as templates to evaluate the specificity of the developed one-step multiplex RT-qPCR. However, it is pity that we did not calculate the exact concentrations of these DNA/RNA templates.

 

Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance. 

 

Author’s response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

 

The authors should include this statement in their materials and methods and refer to the results from all combinations in the supplemental data. The sensitivity at lower genome copies in the context of single vs co-infections may vary.

Response: We agree to the reviewer’s suggestion. In the pre-experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Due to the limitation of the length of the manuscript, we did not provide the data in this manuscript. Please see Lines 373-378 in the revised manuscript.

 

Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

 

The figure caption for panels A-D shows Cycle (Ct) values on the horizontal-axis, while panel E shows Cycle (Ct) values on the vertical-axis. Please make this distinction in the legend. Furthermore, increase the panel sizes for A-D to match E. The font sizes and figure labels are oddly placed inside the plot. Please place it in the top upper left corner outside of each plot.

Response: Figure 2 has been revised according to the reviewer’s suggestion. The figure labels have been removed to the top upper left corner outside of each plot. The font sizes have been corrected. The figure caption for panels A-D shows Cycle (Ct) values on the horizontal-axis, while panel E shows Cycle (Ct) values on the vertical-axis. This is correct. Please see Figure 2 in the revised manuscript.

 

 

Reviewer #4 (Round 2)

 

Comments and Suggestions for Authors

All the concerns are well addressed; I have one add-up: there is a similar paper dealing with multiplex qPCR detection of various pathogens (10.1016/j.talanta.2024.126351), which could be cited in this manuscript. 

Response: We agree to the reviewer’s suggestion. This paper has been added as a reference in the revised manuscript. Please see Reference 40 in the revised manuscript.

 

Comments on the Quality of English Language

should be improved appropriately.

Response: We agree to the reviewer’s suggestion. This manuscript has been proofread by MDPI (No. english-85908). Please see the revised manuscript.

 

Best regards,

 

Kaichuang Shi

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

All the concerns are well addressed; I have one add-up: there is a similar paper dealing with multiplex qPCR detection of various pathogens (10.1016/j.talanta.2024.126351), which could be cited in this manuscript. 

Comments on the Quality of English Language

should be improved appropriately.

Author Response

The Cover Letter

October 12, 2024

 

Dear editor,

Our manuscript has been revised carefully according to the reviewers’ suggestions. The details are follows.

 

Reviewer #2 (Round 2)

 

Comments and Suggestions for Authors

The revised manuscript is improved in quality due to sincere efforts from the authors. The discussion section is significantly better. A new comment to this revised version and responses to the author’s responses are included below.

 

Major concern (version 2)

 

One gold-standard to demonstrate PCR assay sensitivity, is to ensure whether the primers are amplifying the correct target. Can the authors sequence the amplicons that were generated from clinical samples, perform multiple sequence alignment and BLAST analysis to confirm the specificity of the primers? This would make the assay specificity argument more compelling. 

Response: We agree to the reviewer’s suggestion. This job has been done during our experiments. In this study, a total of 1869 clinical samples were tested using the developed quadruplex RT-qPCR, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93% (36/1869), 9.36% (175/1869), 0.32% (6/1869), and 0.75% (14/1869), respectively. Of these positive samples, we selected some positive samples to amplify and sequence the target fragments, and performed multiple sequence alignment and BLAST analysis to confirm that the amplified fragments were correct. This content has been added in the revised manuscript. Please see Lines 321-325 in the revised manuscript.

 

Major concerns (version 1)

 

Major concern 1: The fundamental methodology is somewhat flawed and requires more rigor – all analysis of sensitivity and specificity were performed with purified FeKoV, FeAstV, and FRV target DNA templates when they should have been performed using RNA. After all, it is a RT-qPCR assay. FeBuV is a DNA virus, so it can be argued that the current methodology is more appropriate, although in practice, the authors would want to detect FeBuV RNA present in clinical samples. Doing so will accurately estimate the “RT” i.e., the cDNA synthesis efficiency. This should accurately reflect the viral RNA that will be present in clinical samples. One option for the authors is to in vitro transcribe viral RNA from p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV by adding a simple upstream T7/SP6 promoter. The resulting RNA should be subjected to the same purification and cDNA synthesis steps as clinical samples. 

 

Author’s response: In the experiments for sensitivity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluate the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. In the experiments for specificity analysis, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, and the positive clinical samples of FeKoV, FeAstV, FeBuV, and FRV were used as positive controls to validate the specificity of the developed quadruplex RT-qPCR assay. Since the quadruplex RT-qPCR was one-step RT-qPCR, the steps of reversed transcription of RNA to cDNA, and amplification of PCR based on cDNA/DNA were continuously performed in one tube. Therefore, the nucleic acids (RNA/DNA) were used as templates directly to amplify the target fragment using the developed quadruplex RT-qPCR assay. We agree to the reviewer’s suggestion. The developed quadruplex RT-qPCR was one-step RT-qPCR, so the nucleic acids (RNA/DNA) were used as templates directly to test FeKoV, FeAstV, FeBuV, and FRV. The steps of reversed transcription, and amplification were continuously performed in one tube. At present, the recombinant plasmids which contain the target gene fragment, and the recombinant plasmids which contain the cRNA of target fragment are commonly used as the control standards for RT-qPCR/qPCR. Two kinds of them are acceptable. Therefore, the recombinant plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV, which contain the gene fragments of FeKoV, FeAstV, FeBuV, and FRV, respectively, were used as standard plasmids for development of the quadruplex RT-qPCR assay in this study.

 

The fact that the authors use a One-step RT PCR setup does not exclude the variability introduced in sample purification and RT-steps. Can the authors confirm whether the assay sensitivity is comparable between the plasmid vs RNA templates? 

Response: In this study, the recombinant standard plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were used to evaluate the limits of detections (LODs) of the developed quadruplex RT-qPCR assay. We are sorry that we did not use RNA to analyze the assay’s sensitivity, so we could not compare the results between plasmids and RNA templates in this study.

 

Major concern 2: The specificity analysis section is lacking in detail – especially given its importance to the overall study. How many copies of the “contaminating” viral templates were used? Have the authors tested a range of dilutions? 

 

Author’s response: We agree to the reviewer’s suggestion. The four plasmid constructs p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV were mixed and diluted 10-fold serially, and the mixture with 10⁸ copies/μL of each plasmid were used as positive control templates to evaluate the specificity of the developed quadruplex RT-qPCR assay. In addition, the total nucleic acids were extracted from the positive samples of FeKoV, FeAstV, FeBuV, and FRV, and used as positive controls for the developed quadruplex RT-qPCR assay. This has been added in the revised manuscript. Please see Line 179 in the revised manuscript.

 

The author’s response and revision in the text does not address the original question. The specificity can only really be tested by using varying concentrations of target (FeKoV, FeAstV, FeBuV, and FRV) and non-target/contaminating (FPV, FCoV, FBoV, FCV, and FHV) template. In the absence of such data, it is unclear whether or not this assay can detect FeKoV, FeAstV, FeBuV, and FRV in clinical samples that have varying loads of FPV, FCoV, FBoV, FCV, and FHV.

Response: We agree to the reviewer’s suggestion. The vaccine solutions of FPV, FHV, and FCV, and the positive clinical samples of FCoV, and FBoV were used to extracted the total nucleic acids (DNA/RNA), and used as templates to evaluate the specificity of the developed one-step multiplex RT-qPCR. However, it is pity that we did not calculate the exact concentrations of these DNA/RNA templates.

 

Major concern 3: The sensitivity analysis method requires more rigor. For instance, the authors use purified plasmids of p-FeKoV, p-FeAstV, p-FeBuV, and p-FRV mixed at an equal ratio of 1:1:1:1 to generate standard curves. Then for the sensitivity analysis, diluted this mixture to test. Why are all the templates present at equal copies? Surely during instances of natural infection and co-infections, viral loads for each viral species will vary – often by orders of magnitude. To properly judge sensitivity, the authors should test combinations where one or more of the viral templates are present at higher abundance. 

 

Author’s response: We agree to the reviewer’s suggestion. Surely, during instances of natural infection and co-infections, viral loads for each viral species will vary. It is impossible that the viral loads of different viruses in the co-infected animals are equal to each other, and we cannot predict the possibility of various combinations of viral loads. During experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Therefore, we described the result of the equal ratio of 1:1:1:1 to evaluate the sensitivity of the developed quadruplex RT-qPCR assay. Please see Lines 267-274 in the revised manuscript.

 

The authors should include this statement in their materials and methods and refer to the results from all combinations in the supplemental data. The sensitivity at lower genome copies in the context of single vs co-infections may vary.

Response: We agree to the reviewer’s suggestion. In the pre-experiments, we tried different combinations of viral loads, and equal loads was one of these combinations. Due to the limitation of the length of the manuscript, we did not provide the data in this manuscript. Please see Lines 373-378 in the revised manuscript.

 

Minor comment 2: The figure panel letters are missing from A-D. Please also change the X-axis label for panel to reflect genome copies/(mass of DNA/RNA).

 

The figure caption for panels A-D shows Cycle (Ct) values on the horizontal-axis, while panel E shows Cycle (Ct) values on the vertical-axis. Please make this distinction in the legend. Furthermore, increase the panel sizes for A-D to match E. The font sizes and figure labels are oddly placed inside the plot. Please place it in the top upper left corner outside of each plot.

Response: Figure 2 has been revised according to the reviewer’s suggestion. The figure labels have been removed to the top upper left corner outside of each plot. The font sizes have been corrected. The figure caption for panels A-D shows Cycle (Ct) values on the horizontal-axis, while panel E shows Cycle (Ct) values on the vertical-axis. This is correct. Please see Figure 2 in the revised manuscript.

 

 

Reviewer #4 (Round 2)

 

Comments and Suggestions for Authors

All the concerns are well addressed; I have one add-up: there is a similar paper dealing with multiplex qPCR detection of various pathogens (10.1016/j.talanta.2024.126351), which could be cited in this manuscript. 

Response: We agree to the reviewer’s suggestion. This paper has been added as a reference in the revised manuscript. Please see Reference 40 in the revised manuscript.

 

Comments on the Quality of English Language

should be improved appropriately.

Response: We agree to the reviewer’s suggestion. This manuscript has been proofread by MDPI (No. english-85908). Please see the revised manuscript.

 

Best regards,

 

Kaichuang Shi

 

Author Response File: Author Response.pdf