Review Reports - A Recombinant Porcine Epidemic Diarrhea Virus with Multiple S2 Subunit Mutations from China: Isolation, Genetic Characterization, and Pathogenicity Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer’s Comments to Authors:

Abstract:

Line 20: Use the word ‘experimental’ prior to intestinal sample.
Line 21: correct as complete instead of compete
Line 23: Inconsistent strain name as PEDV/FJLY202201. Kindly check the PEDV strain. Both FJLY2021 and FJLY202201 are used in the text.
Line 28: Use ‘is’ instead of ‘was’
Further, check all the repeat sentences, spelling mistakes, spacing error etc. throughout the text.

Introduction:

Insufficient previous data
Lack of clinical sign and pathological lesion data
More scientific phrasing is recommended.
Line 46: It is better to use single- stranded RNA virus
Line 46 to 49: Incomplete or inaccurate sentence
Line 50 to 53: The sentence is too long and complex.

Materials and Methods:

Line 75: write a brief procedure how samples were collected? From pig/ piglets? Mention carefully.
Mention the source of Vero cell
Mention the detail of fluorescence microscope
Line 77- Duplication in the sentence
Line 80- Specify the quantity of inoculum used
Line 84 to 88- Lacks clarity. The method of purification is not explained.
Line 93- use supernatant
The washing steps in the immunofluorescence procedure are not mentioned. Also, the DAPI staining is to be performed at room temperature.
Line 130- correct as ventilation. It is recommended to rewrite the sentence for better clarity.
Justification for the route and volume of inoculation: The experimental group was inoculated via the intraoral route to closely mimic the natural mode of PEDV infection through the gastrointestinal tract, thereby ensuring accurate assessment of pathogenicity. In contrast, the control group received intranasal inoculation to serve as a non-enteric exposure model. However, the reported intranasal volume of 3 mL appears excessive for neonatal piglets and may not be physiologically appropriate.
Line 132: Justify intranasal DMEM inoculation.
Line 136: rephrase the sentence
Line 136 to 138 - Needs more specificity and clarity
Line 137: provide the euthanasia protocol
Line 139: use ‘spread over a sheet’ instead of ‘separated’
Line 139- Rewrite the sentence for better understanding.
Line 141: provide brief IHC protocol
The challenge group animals were inoculated orally with virus but the control group animals were inoculated intranasally. Authors are requested to justify these differences in routes.
The authors have mentioned some places as PEDV/FJLY2021 and some places as PEDV/FJLY202201. The authors are requested to correct the strain discrepancies PEDV/FJLY2021 and PEDV/FJLY202201. This discrepancy should be corrected throughout the manuscript.
The authors are requested to mention the biosafety protocols followed while handling this highly virulent pathogen.
The authors are requested to mention the containment level and risk mitigation measures.

Results:

Line 173-176- The structural genes to be listed promptly
Line 202- The breakpoint occurs at a single nucleotide position. A breakpoint cannot be simultaneously in S1 and S2 at the exact same nucleotide
Use italics for amino acid changes
Line 281- Please explain what lysis in this context mean?
Spelling correction- Duodenum, Jejunum, Ileum
Rephrase the legends of Figure 1, as there is ambiguity
Sub-head 3.5.: clinical signs, gross and histopathology can be presented in tabular form for better clarity
Figure 3 is missing in the text. The authors are requested to mention.
Figure 5 is unsatisfactory as it lacks proper annotation like arrow, arrowheads to etc specify any particular lesion. Also, the legends are insufficient. Please modify accordingly

Discussion:

Line 302- 305- Overly long and complex sentences reduced the clarity of expression
Line 343- The ORF is the frame, not the protein itself
Previous references mentioning clinical aspects, gross and histopathology should be included to support the present data

Conclusion:

Line 364-368 -strengthen the concluding sentence to directly address the global threat mentioned in the introduction.
Lines 26 & 362: justify ‘never been reported before’ and ‘rarely reported’

References

Insufficient
Please write/ organise all the references according to the journal protocol and make it uniform.
Numerous grammatical errors are there throughout the manuscript.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Need significant improvement. The written English is moderately standard. Few sentences need clarity with appropriate words by reframing the sentences. In addition, typographical and grammatical errors are present in the manuscript, which need to be rectified.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This work submitted by Yan et al. reports the identification of a new Porcine Epidemic Diarrhea Virus variant named PEDV/FJLY2021 from the Fujian region in China. Briefly, the authors sequenced the whole genome of a diarrheal piglet and compared it with other typical diarrhea-related viruses (PEDV, PEAV, TGEV, PDCoV). They identified that this virus belongs to PEDV and exhibits typical cytopathic effects along with other related features confirmed through PCR, immunostaining, and electron microscopy. Whole-genome comparison and phylogenetic analysis revealed that this new strain falls under the G2a subgroup and contains three recombination events—one in ORF1b and two in the S protein. Further analysis identified three major mutations in the S protein that may confer pathogenicity.

Importantly, pathogenicity was confirmed by orally administering DMEM or the PEDV/FJLY202201 strain to 3-day-old piglets. Clinical symptoms, including diarrhea, vomiting, and anorexia, were observed only in the PEDV/FJLY202201-infected group and not in the DMEM control group. Histopathological analysis of the duodenum, jejunum, and ileum further supported these findings.

Overall, this is a scientifically sound manuscript describing the identification of a new PEDV strain (PEDV/FJLY202201), confirmed through genetic, bioinformatic, and microscopic analyses. One major issue with this manuscript is the presence of typographical errors in the figure panels, which do not align with the text, along with several missing technical and scientific details. Please see my comments below:

a. Figure 1C: It is unclear what the two rows represent. Are these mock vs. infected samples? Also, no details are provided about the type of stain used for PEDV. Was an antibody used?

b. Figure 3B: I recommend showing only significant mutations. Also indicate that the numbers at the bottom represent amino acids (aa), and highlight only important breakpoints—for example, the start and end of the SS2 region (aa 748–755). The current presentation is vague. I also strongly recommend abbreviating COE, SS2, SS6, and 2C10 in the legend wherever appropriate.

c. Figure 5A: All panels contain typographical errors: duodcnum, Jcjunum, ilium. Please correct all of these.

d. Clearly indicate what the subsections (b–g) represent in the legend for Figure 5.

e. For all figures, include the number of experimental repeats and any statistical analyses performed. This information should also be included in the Methods section.

f. Abbreviate all technical terms when possible. Several abbreviations such as COE and IHC are missing.

Comments on the Quality of English Language

Lots of grammatical errors

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Lane 34, change the word "symptoms" to "clinical signs," and do the same throughout the document.

Lane 35, correct: the first clinical description was made in 1971 in the UK; the first isolation was later.

Lanes 37-39, briefly describe the characteristics of PEDV from those 2010 outbreaks. What does "new variant" mean?

Lanes 76-78, repeated phrase (These samples were diluted with an equal amount of phosphate-76 buffered saline (PBS)).

Table 1, include the genotype of each reference strain.

Lane 131, indicate the passage number used in the inoculation.

Lanes 140-142, indicate the brand of the mAb used.

Figure 1: In the IFA image, a single infected cell is observed, while under the microscope, a very abundant cytopathic effect is identified. Explain in the document why this labeling is so low. If there is no explanation, modify the figure and show a photograph where greater labeling is identified.

For the analysis of potential recombination events, why are reference strains used, including those that are not from your country? That is, are these strains likely to be present in the territory where the study was conducted? The intention of the analysis seems poorly defined. If possible, include part of this in the discussion.

Figure 4. The identifiers in the text are incorrect.

It's surprising that the Mock group didn't gain weight during the study period (7 days). What type of diet did they offer? Please mention this in M&M.

Was viral load (qRT-PCR) not assessed in stool and digestive tract organs? What was the reason for this?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have carefully reviewed the revised manuscript and the authors’ responses. All reviewer comments have been addressed comprehensively, and the authors have made thoughtful and meticulous revisions to enhance the manuscript.

Author Response

Dear reviewer,

Thank you for your kind message and positive assessment of our revised manuscript. We are very pleased to know that the changes met your expectations. We appreciate your guidance throughout the review process.