Development of an Indirect ELISA to Distinguish between Porcine Sapelovirus-Infected and -Vaccinated Animals Using the Viral Nonstructural Protein 3AB

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the PSV 3AB protein was expressed and purified by a prokaryotic expression system, and an indirect ELISA method was established for the differential diagnosis of PSV-infected and vaccine-immunized animals. However, there are some points to be considered,

1. Please double check the titles of section 3.2 and 3.3 in the results.

2. Were replicated tests performed in the results sections of 3.2 and 3.4, and why were there no t-tests?

3. Why did the authors choose to use a specific positive determination value rather than the specimen/negative control value (S/N value) for positive determination in the determination of results?

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I have read the manuscript "Development of an indirect ELISA to distinguish between porcine sapelovirus-infected and -vaccinated animals using the viral nonstructural protein 3AB,"   and found it interesting. However, the following comments need to be addressed before its being considered for publication.

 

General comments

Writing Style: Needs careful proofreading to correct minor grammatical errors and improve the overall flow of the text.

Specific comments

1. Abstract and Introduction:
• Line 67: what is i-ELISA?
• The sentence in Line 68 needs revision.
2. Methods: The methodology section is thorough but somewhat disorganised. Key details such as the selection criteria for serum samples (e.g., number, health status, and geographical origin of pigs) need more explicit explanation to ensure reproducibility and validate the results. The authors mention that serum samples were collected from cattle (line 73), which appears to be an error, as the study focuses on pigs. Such inaccuracies can undermine the credibility of the study. Moreover, the manuscript could benefit from a clearer explanation of the statistical methods used, especially concerning the ROC curve analysis, to enhance the rigor of the study.

·        True positive and true negative samples are required to analyse ROC curve and determine the optimal coating antigen concentration and serum dilution. In the methods section, it's unclear how the samples used as a true negative and true positive are selected or evaluated.

·        Assay sensitivity refers to the ability of a diagnostic test or assay to correctly identify individuals who have the disease or condition in question. It is the proportion of true positives (those who actually have the virus) that are correctly identified by the test. However, the method used to carry out assay sensitivity in this manuscript is quite different. Please consider revising.

·        For Assay specificity, in addition to what has already been described, it would be great if the authors try to add some analysis like adding some data on the proportion of true negatives (those who do not have the virus) that are correctly identified by the test.

3. Results:

·        How much is high? While the study reports high sensitivity and specificity, it does not indicate the actual numeric values of the assay sensitivity and specificity. Please revise the methods so that you have a numeric value to explain the exact sensitivity and specificity of the assay.

·        Line 657: the word antigencity might not be correctly used in this situation; please consider revision.

·         

·        Section 3.2. Optimisation of the indirect ELISA protocol and Section 3.3. The indirect ELISA protocol is optimised and duplicated. Both paragraphs are duplicates of each other and describe the methods, not the results. Please remove section 3.3. and then update section 3.2 by changing the results and moving the contents that look like methods to the method section.

·        Reproducibility and repeatability refer to the same thing. However, both terms are used in the current manuscript. Please be consistent with the words that you use; either use reproducibility or repeatability.

4. Discussion:

·        The discussion is generic, lacks coherence and does not adequately address the broader implications of the findings. For instance, the potential for cross-reactivity with other non-PSV picornaviruses should be explored. The discussion should also critically assess the limitations of the study, such as the small sample size and the geographical limitation of the serum samples, which may affect the generalizability of the findings.

·        Line 261:  the phrase “Authors In this study..” does not seem important here.

 

·        Many parts of the discussion lack citation.

Comments on the Quality of English Language

Some of the paragraphs contain unnecessary words or phrases and some grammatical errors that need to be corrected.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf