Extrahepatic Replication Sites of Hepatitis E Virus (HEV)

Round 1

Reviewer 1 Report

Manuscript Number: zoonoticdis-2161564

Title: Extrahepatic Replication Sites of Hepatitis E virus (HEV)

The review article provides information about the extrahepatic replication sites for HEV. The article presents an exhaustive list of the literature containing HEV-related disorders observed in humans and the animal models used to study some of these disorders/conditions. However, there are major and minor aspects that need to be addressed to improve the article quality so that it is more informative to the reader.

Major comments:

1.       Please combine all the rarely reported body systems from 2.4 Endocrine System through 2.9 Genital (Reproductive) System under one title, e.g. “2.4. Other body systems”. There is no much reported about the involvement of HEV infection in these systems, and thus no need to make separate subtitle for each system. Also, please remove tables 4 through 9 as most of these tables have only one condition and no need to use a table for that. Instead, mention the condition and cite the reference immediately after it. Here is an example for 2.4. Endocrine System: you can write smoothing like “An endocrine system disorder, autoimmune thyroiditis, was reported in a middle-aged woman with acute hepatitis E virus infection (76).”  This can be applied to all systems from 2.4 to 2.9.

2.       The author made a list of the body systems involved at some level in HEV infections, without giving any more details. Please elaborate on how HEV infection is involved in the disorders cited. There should be at least a few sentences giving some details about the condition or the circumstances/context where the HEV infection is related to that condition. For example, in 2.9. Genital/reproductive System: the author should mention some of the findings from the cited reference #82; something like: “In that study, 28.11% (52/185) of the infertile males from one city were HEV RNA positive, with higher prevalence of HEV in semen compared to serum of pregnant women or the general population. Also, the author may comment on the results from the semen analysis of HEV-infected infertile males and that it was found in some patients that the blood–testis barrier was destroyed by HEV infection. Another examples, please clarify how the immune system was affected by the HEV infection leading to “Acute Graft Dysfunction” (Ref #81), providing some context. It’s reported that this condition happened 17 years after a liver transplant. Please give more details and how the immune system was involved, so that the reader gets more informed.

3.       The author needs also to elaborate on the HEV infection and the associated effects on 2.1. Nervous and Musculoskeletal System, 2.2. Cardiovascular System, and 2.3. Digestive System.

Specific Comments:

1.       I wonder why on the manuscript footnotes it shows “Acoustics 2022”, not “zoonotic diseases”? This may need to be corrected by the editorial office.  

2.       Line 9: Replace "blood testes" with "blood-testis".

3.       Line 9-10: Remove "This recent and other" and start the sentence with Extrahepatic ....."

4.       Line 11-12: Change "All these extrahepatic diseases are very important" to "Knowledge about HEV-related extrahepatic diseases is very important......"

5.       Line 12: Replace "a clinician" with "clinicians".

6.       Line 15: Replace "a sporadic outbreak" with to "sporadic outbreaks"

7.       line 24: Replace "genital" with "reproductive"; also replace "genital system" with "reproductive system" anywhere in the article.

8.       Line 24-25: Replace "All these extrahepatic signs are aided by..." to "All extrahepatic signs are caused by..."

9.       Line 27: Replace "blood brain" with "blood-brain". Line 27: Replace "blood testes" with "blood-testis"

10.   Line 40-41: "Orthohepevirinae and Parahepevirinae" subfamilies should be in italics"

11.   Line 42: "Orthohepevirinae" should be in italics. Replace "comprising of" to "consisting of".

12.   Line 43: Replace "are due to Paslahepevirus balayani" with "belong to species Paslahepevirus balayani". Also, the species name "Paslahepevirus balayani" should be in italics.

13.   Line 44: Replace (gt1, gt2, gt3 and gt4) with (gt1-gt4).

14.   Line 44: Please cite this official classification paper PMID: 36170152, in addition to reference #4.

15.   Line 53: Replace "Hepatitis E virus [HEV]" with "HEV" only since this acronym was explained earlier.

16.   Line 54: Replace "It comprises of" with "It is comprised of".

17.   Line 55: Replace "also comprise of" to "also contain". Line 55: Replace "biggest" with "largest".

18.   Line 58: Add a comma "," after "viroporin".

19.   Line 61: Replace "has" with "have".

20.   Line 63: "in vitro" and "in vivo" should be in italics.

21.   Line 73: Replace "There exists lot of loopholes" with "There are lots of loopholes".

22.   Line 78: "in vitro" should be in italics.

23.   Line 92: Replace "capable of HEV replication." with "where HEV replication occurs."

24.   Line 104: Replace "attributable" with "attributed".

25.   Line 116-117: Replace "while HEV infection" with "while having an HEV infection"

26.   Line 148: Change the numbering of the subtitle from "2.10." to "3." as it's irrelevant to the previous list of human systems.

27.   Line 150: "in vitro" should be in italics.

28.   Line 152: Replace "Till date" with "Till now"

29.   Line 160: Replace "HEV used" with "HEV genotypes and/or strains used".

30.   Line 163: Change the numbering of the subtitle from "2.11." to "4." as it discusses a new, independent section.

31.   Line 169-171: Please rephrase the following sentence as it is not very clear: "Genetic plasticity is one of the advantageous attributes of RNA viruses, thus, they can promptly produce drug-resistant viral populations helping to escape the immune system of host while checked homeostatic pressure."

32.   Line 183-185: Please rephrase the following sentence as it is not very clear: "Reports of acute HEV hepatitis patients consisting of genomic rearrangements were identified and further demonstrate the association of HEV persistence and ribavirin insensitivity with enhanced population heterogeneity"

33.   Line 202: "in vitro" and "in vivo" should be in italics.

34.   Line 206-207: References #82 (Huang at al 2018) and #128 (Horvatits at al 2021) should be added at the end of this sentence starting with "In addition, HEV is shown ....".

35.   Line 209: Replace “blood testes barrier” with “blood-testis barrier”. Remove references #82 and 128, and replace with a relevant reference, such as #125.

36.   Line 215: Correct "zoonoses" to "zoonosis".

37.   Line 219: Change the numbering of the subtitle from "3." to "5." based on the new suggestions.

38.   There are many references missing the page (or other) information, please complete this missing info to be consistent with the reset of the references list. Examples are #1, 2, 4, 10, 11, 14, 23, 24, 47, etc. 

END

Author Response

We would like to thank the editor to go through our manuscript “Extrahepatic replication sites of Hepatitis E virus (HEV)” and consider to be applicable for the “zoonotic diseases”.

We would like to thank the reviewers for their feedback that helped us to improve our manuscript. We have addressed every comment from the reviewers and revised our manuscript accordingly. Please find our edits and responses to the reviewer comments below.

 

 

Reviewer 1:

 

The review article provides information about the extrahepatic replication sites for HEV. The article presents an exhaustive list of the literature containing HEV-related disorders observed in humans and the animal models used to study some of these disorders/conditions. However, there are major and minor aspects that need to be addressed to improve the article quality so that it is more informative to the reader.

Major comments:

1. Please combine all the rarely reported body systems from 2.4 Endocrine System through 2.9 Genital (Reproductive) System under one title, e.g. “4. Other body systems”. There is no much reported about the involvement of HEV infection in these systems, and thus no need to make separate subtitle for each system. Also, please remove tables 4 through 9 as most of these tables have only one condition and no need to use a table for that. Instead, mention the condition and cite the reference immediately after it. Here is an example for 2.4. Endocrine System: you can write smoothing like “An endocrine system disorder, autoimmune thyroiditis, was reported in a middle-aged woman with acute hepatitis E virus infection (76).”  This can be applied to all systems from 2.4 to 2.9.

Answer: Thank you for your feedback. We have combined the rarely reported body system and have added the information as suggested by the reviewer, line 169 – 255.

2. The author made a list of the body systems involved at some level in HEV infections, without giving any more details V infection is related to that condition. For example, in 2.9. Genital/reproductive System:the author should mention some of the findings from the cited reference #82; something like: “In that study, 28.11% (52/185) of the infertile males from one city were HEV RNA positive, with higher prevalence of HEV in semen compared to serum of pregnant women or the general population. Also, the author may comment on the results from the semen analysis of HEV-infected infertile males and that it was found in some patients that the blood–testis barrier was destroyed by HEV infection. Another examples, please clarify how the immune system was affected by the HEV infection leading to “Acute Graft Dysfunction” (Ref #81), providing some context. It’s reported that this condition happened 17 years after a liver transplant. Please give more details and how the immune system was involved, so that the reader gets more informed.

Answer: We would like to thank the reviewer for this wonderful suggestion. We have added some details related to the clinical disorder, line 169 -255; line 247`-253.

 

3. The author needs also to elaborate on the HEV infection and the associated effects on 2.1. Nervous and Musculoskeletal System, 2.2. Cardiovascular System, and 2.3. Digestive System.

Answer: We would like to thank the reviewer for suggesting this idea. We have added the details as suggested by the reviewer, line 169-255.

Specific Comments:

1. I wonder why on the manuscript footnotes it shows “Acoustics 2022”, not “zoonotic diseases”? This may need to be corrected by the editorial office.  

Answer: Editorial office can answer that!

2. Line 9: Replace "blood testes" with "blood-testis".

Answer: Changes made!

3. Line 9-10: Remove "This recent and other" and start the sentence with Extrahepatic ....."

Answer: Changes made, line 8.

 

4. Line 11-12: Change "All these extrahepatic diseases are very important" to "Knowledge about HEV-related extrahepatic diseases is very important......"

Answer: Changes made, line 10.

5. Line 12: Replace "a clinician" with "clinicians".

Answer: Changes made, line 11.

6. Line 15: Replace "a sporadic outbreak" with to "sporadic outbreaks"

Answer: Changes made, line 14.

7. line 24: Replace "genital" with "reproductive"; also replace "genital system" with "reproductive system" anywhere in the article.

Answer: Changes made.

8. Line 24-25: Replace "All these extrahepatic signs are aided by..." to "All extrahepatic signs are caused by..."

Answer: Changes made, line 27.

9. Line 27: Replace "blood brain" with "blood-brain". Line 27: Replace "blood testes" with "blood-testis"

Answer: Changes made.

10. Line 40-41: "Orthohepevirinae and Parahepevirinae" subfamilies should be in italics"

Answer: Changes made, line 44.

11. Line 42: "Orthohepevirinae" should be in italics. Replace "comprising of" to "consisting of".

Answer: Changes made, line 45.

12. Line 43: Replace "are due to Paslahepevirus balayani" with "belong to species Paslahepevirus balayani". Also, the species name "Paslahepevirus balayani" should be in italics.

Answer: Changes made, line 46.

13. Line 44: Replace (gt1, gt2, gt3 and gt4) with (gt1-gt4).

Answer: Changes made, line 48.

14. Line 44: Please cite this official classification paper PMID: 36170152, in addition to reference #4.

Answer: Changes made, line 48.

15. Line 53: Replace "Hepatitis E virus [HEV]" with "HEV" only since this acronym was explained earlier.

Answer: Changes made, line 57.

16. Line 54: Replace "It comprises of" with "It is comprised of".

Answer: Changes made, line 57,58.

17. Line 55: Replace "also comprise of" to "also contain". Line 55: Replace "biggest" with "largest".

Answer: Changes made, line 59.

18. 18.   Line 58: Add a comma "," after "viroporin".

Answer: Changes made, line 62.

19. 19.   Line 61: Replace "has" with "have".

Answer: Changes made, line 65.

20. 20.   Line 63: "in vitro" and "in vivo" should be in italics.

Answer: Changes made, line 68.                          

21. Line 73: Replace "There exists lot of loopholes" with "There are lots of loopholes".

Answer: Changes made, line 130.

22. 22.   Line 78: "in vitro" should be in italics.

Answer: Changes made, line 135.

23. 23.   Line 92: Replace "capable of HEV replication." with "where HEV replication occurs."

Answer: Changes made, line 150.

24. 24.   Line 104: Replace "attributable" with "attributed".

Answer: Changes made, line 164.

25. Line 116-117: Replace "while HEV infection" with "while having an HEV infection"

Answer: Changes made, line 176.

26. 26.   Line 148: Change the numbering of the subtitle from "2.10." to "3." as it's irrelevant to the previous list of human systems.

Answer: Changes made.

27. Line 150: "in vitro" should be in italics.

Answer: Changes made. All “in vitro” and “in vivo” are mentioned in italics.

28. 28.   Line 152: Replace "Till date" with "Till now"

Answer: Changes made, line 260.

29. Line 160: Replace "HEV used" with "HEV genotypes and/or strains used".

Answer: Changes made, line 269.

30. 30.   Line 163: Change the numbering of the subtitle from "2.11." to "4." as it discusses a new, independent section.

Answer: Changes made, line 274.

31. Line 169-171: Please rephrase the following sentence as it is not very clear: "Genetic plasticity is one of the advantageous attributes of RNA viruses, thus, they can promptly produce drug-resistant viral populations helping to escape the immune system of host while checked homeostatic pressure."

Answer: Changes made, line 280-283.

32. Line 183-185: Please rephrase the following sentence as it is not very clear: "Reports of acute HEV hepatitis patients consisting of genomic rearrangements were identified and further demonstrate the association of HEV persistence and ribavirin insensitivity with enhanced population heterogeneity"

Answer: Changes made, line 295-298.

33. Line 202: "in vitro" and "in vivo" should be in italics.

Answer: Changes made. All “in vitro” and “in vivo” are mentioned in italics.

 

34. Line 206-207: References #82 (Huang at al 2018) and #128 (Horvatits at al 2021) should be added at the end of this sentence starting with "In addition, HEV is shown ....".

Answer: Changes made, line 320.

35. 35.   Line 209: Replace “blood testes barrier” with “blood-testis barrier”. Remove references #82 and 128, and replace with a relevant reference, such as #125.

Answer: Changes made, line 322. We added the reference suggested by the reviewer. However, we could not remove the references as the reference discussed the differences between the HEV particles in the serum and semen. The other reference is referring to the ability to cross blood-testis barrier.

36. 36.   Line 215: Correct "zoonoses" to "zoonosis".

Answer: Changes made, line 329.

37. Line 219: Change the numbering of the subtitle from "3." to "5." based on the new suggestions.

Answer: Changes made.

38. There are many references missing the page (or other) information, please complete this missing info to be consistent with the reset of the references list. Examples are #1, 2, 4, 10, 11, 14, 23, 24, 47, etc.

Answer: We are sorry to inform you that the references you have mentioned are from the sources where we could not locate the page numbers to include in these references.

Reviewer 2 Report

Although many excellent review articles on extrahepatic manifestations and replication sites of hepatitis E virus (HEV) have thus far been published (Gastroenterol Rep 4: 1-15, 2016; Liver Int 36: 467-472, 2016; J Hepatol 66: 1082-1095, 2017; Cures 11: e5499, 2019; Rev Med Virol 31: e2218, 2021), this article comprehensively reviews the published literatures regarding the body systems related to clinical diseases and multiple animal models that have been used to investigate the extrahepatic replication sites of HEV. This article updates our understanding on the extrahepatic manifestations and replication sites of HEV and seems to be worthy of publication. However, there are several concerns that need to be addressed as described below.

 

Comments:

1.      In accordance with the title of this article, the extrahepatic replication sites of HEV should be summarized first (before section 2), accompanied by evidence demonstrating viral replication in each site, such as “proven by a negative-strand-specific reverse transcriptase PCR” or “proven by in situ hybridization”, since the mere presence of HEV RNA does not provide evidence of HEV replication in the examined tissues and it is described in Abstract (line 20-21) that virus replication has been demonstrated in multiple organs.

2.      The number of patients in Figures 1-3 can be incorporated into Tables 1-3, respectively. In addition, Tables 4-9 should include the number of patients.

3.      The Table between line 162 and 163 need Table no. and title.

4.      Subsection 2.11 (Notable Abilities of HEV) should be removed, since the descriptions in this subsection are unrelated to this review article.  

5.      Line 17: Please specify “produce genotype specific lesions” in the text.

6.      Line 44 should include gt7.

7.      Line 57: “123” should be corrected to “112-114”.

8.      Line 70 needs relevant references.

9.      There are many typographical and grammatical errors throughout the manuscript.

10.  Some references (1, 2, 4, 14, 23, 47, 52, 57, 59, 76, 102, 115, and 131) are incomplete.

Author Response

Reviewer 2:

Although many excellent review articles on extrahepatic manifestations and replication sites of hepatitis E virus (HEV) have thus far been published (Gastroenterol Rep 4: 1-15, 2016; Liver Int 36: 467-472, 2016; J Hepatol 66: 1082-1095, 2017; Cures 11: e5499, 2019; Rev Med Virol 31: e2218, 2021), this article comprehensively reviews the published literatures regarding the body systems related to clinical diseases and multiple animal models that have been used to investigate the extrahepatic replication sites of HEV. This article updates our understanding on the extrahepatic manifestations and replication sites of HEV and seems to be worthy of publication. However, there are several concerns that need to be addressed as described below.

Answer: We would like to thank the reviewer for the positive comments.

 

Comments:

1. In accordance with the title of this article, the extrahepatic replication sites of HEV should be summarized first (before section 2), accompanied by evidence demonstrating viral replication in each site, such as “proven by a negative-strand-specific reverse transcriptase PCR” or “proven by in situ hybridization”, since the mere presence of HEV RNA does not provide evidence of HEV replication in the examined tissues and it is described in Abstract (line 20-21) that virus replication has been demonstrated in multiple organs.

Answer: We would like to thank the reviewer for such a wonderful suggestion. We have included separate section “Extrahepatic replicati of HEV” with two sb sections, line 79-158.

2. The number of patients in Figures 1-3 can be incorporated into Tables 1-3, respectively. In addition, Tables 4-9 should include the number of patients.

Answer: No. of patients has been incorporated into tables.

3. The Table between line 162 and 163 need Table no. and title.

Answer: Changes made, line 271.

4. Subsection 2.11 (Notable Abilities of HEV) should be removed, since the descriptions in this subsection are unrelated to this review article.  

Answer: We would like to thank the reviewer for going through our manuscript in detail. We would like to highlight that our manuscript mentions the ability of HEV to cause clinical disorders in 10 of the body systems. Thus, we would like to keep a section suggesting some notable abilities of hepatitis E viruses.

5. Line 17: Please specify “produce genotype specific lesions” in the text.

We would like to thank the reviewer for bringing this point, line 120-127.

“It is very interesting to note that extrahepatic replication related to HEV is not limited to a genotype. From the listed studies, extrahepatic replication in males and non-pregnant females are related to gt3/gt4 HEV. One of the studies reported no evidence of gt1 HEV in the male reproductive system of humans (32). However, an exception was seen when gt1 HEV acute infection was related to the digestive disorder, acalculous cholesystitis (33). HEV gt1 has been related with the female reproductive organs only during the pregnancy. Genotype specific lesions illustrate the need to understand the mechanisms behind the extrahepatic replication of HEV.”

6. Line 44 should include gt7.

Answer: Changes made, line 48.

7. Line 57: “123” should be corrected to “112-114”.

Answer: Changes made, line 61.

8. Line 70 needs relevant references.

Answer: Changes made, line 75.

 

9. There are many typographical and grammatical errors throughout the manuscript.

Answer: We would like to thank the reviewers for bringing this to our notice. We have done detailed revisions to exclude typographical and grammatical errors..

10. Some references (1, 2, 4, 14, 23, 47, 52, 57, 59, 76, 102, 115, and 131) are incomplete.

Answer: We are sorry to inform you that the references you have mentioned are from the sources where we could not locate the page numbers to include in these references.

Round 2

Reviewer 1 Report

In the revised version of the manuscript, the author(s) made a great improvement and addressed all the comments made previously. I recommend the manuscript be accepted for publication after making the minor corrections listed below.

Page 2, second paragraph, line 3 – change “also contain of an ORF4” to “also containing a fourth ORF, ORF4 (12-15)”

Page 2, second paragraph, line 5 – change “sub genomic” to “subgenomic”.

Page 2, section 2.1. – write “ex vivo” in italics.

Page 4, section 3.1.; second paragraph – change “..in one patient” to “in individual cases”.

Page 5, Figure 1. Legend, change “isolated’ to “extracted”.

Page 6, Table 2 and first paragraph, change “Torsade’s” to “Torsades”.

Page 8, section 3.4.; first paragraph – change “membrano proliferative” to “membranoproliferative”.

Page 8, section 3.4.; second paragraph – add “viruses” after “hepatitis E and hepatitis A”.

Page 8, section 3.4.; second paragraph – remove “in the liver transplant patient”.

Page 10, Table 5, continued – no reference is listed for “Pregnant gilts’, in not available, then please remove this row.

Author Response

Reviewer 1:

In the revised version of the manuscript, the author(s) made a great improvement and addressed all the comments made previously. I recommend the manuscript be accepted for publication after making the minor corrections listed below.

Page 2, second paragraph, line 3 – change “also contain of an ORF4” to “also containing a fourth ORF, ORF4 (12-15)”

Answer: Changes made!

Page 2, second paragraph, line 5 – change “sub genomic” to “subgenomic”.

Answer: Changes made!

Page 2, section 2.1. – write “ex vivo” in italics.

Answer: Changes made!

Page 4, section 3.1.; second paragraph – change “..in one patient” to “in individual cases”.

Answer: Changes made!

Page 5, Figure 1. Legend, change “isolated’ to “extracted”.

Answer: Changes made!

Page 6, Table 2 and first paragraph, change “Torsade’s” to “Torsades”.

Answer: Changes made!

Page 8, section 3.4.; first paragraph – change “membrano proliferative” to “membranoproliferative”.

Answer: Changes made!

Page 8, section 3.4.; second paragraph – add “viruses” after “hepatitis E and hepatitis A”.

Answer: Changes made!

Page 8, section 3.4.; second paragraph – remove “in the liver transplant patient”.

Answer: Changes made!

Page 10, Table 5, continued – no reference is listed for “Pregnant gilts’, in not available, then please remove this row.

Answer: Changes made!

Reviewer 2 Report

The manuscript has been revised partly in accordance with my previous comments/suggestions, and the revisions are still unsatisfactory. Therefore, the revised manuscript needs further revisions as described below.

 

Comments:

1.      Since the mere detection of HEV RNA does not indicate actual replication of HEV in the examined tissues, Table 5 should include evidence demonstrating viral replication in each proposed site, such as “proven by a negative-strand-specific reverse transcriptase PCR” or “proven by in situ hybridization”. If such information is not made explicit in the literatures, that in itself is valuable for the readers of this review article and should be provided properly in the table.

2.      In accordance with my previous suggestion, the number of patients in Figures 1-3 has been incorporated into Tables 1-3, respectively. Figures 1-3 with overlapping information with Table 1-3, respectively, have no additional information and should be removed.

3.      Some references (1, 2, 4, 14, 23, 47, 52, 57, 59, 76, 102, 115, and 131) are incomplete. Since they are available from PubMed, they should be completed. For example, the page number of Ref. 1 and Ref. 2 is 331 and 1180, respectively.

Author Response

Reviewer 2:

The manuscript has been revised partly in accordance with my previous comments/suggestions, and the revisions are still unsatisfactory. Therefore, the revised manuscript needs further revisions as described below. 

Answer: We would like to apologize the reviewer if we missed some of the requested revisions. We have tried our best to include every suggestion of the reviewer.

Comments:

1. Since the mere detection of HEV RNA does not indicate actual replication of HEV in the examined tissues, Table 5 should include evidence demonstrating viral replication in each proposed site, such as “proven by a negative-strand-specific reverse transcriptase PCR” or “proven by in situ hybridization”. If such information is not made explicit in the literatures, that in itself is valuable for the readers of this review article and should be provided properly in the table.

Answer: We would like to thank the reviewer for the wonderful suggestion. We have added the column “demonstration of viral replication” in Table 5. As described earlier in section 2.5, the evidence of successful HEV replication requires certain specific assays. We have listed the assays used to prove the extrahepatic replication of HEV.

2. In accordance with my previous suggestion, the number of patients in Figures 1-3 has been incorporated into Tables 1-3, respectively. Figures 1-3 with overlapping information with Table 1-3, respectively, have no additional information and should be removed.

Answer: We completely agree with the reviewer’s concern. However, we had originally included the patient numbers in the figure for easy visualization of the important clinical disorders. We included the patient numbers in the table as per the reviewer request. We strongly believe that a figure visualization has more impact on readers than data presented solely in tabular form. Thus, we would like to keep the figures in the manuscript.

3. Some references (1, 2, 4, 14, 23, 47, 52, 57, 59, 76, 102, 115, and 131) are incomplete. Since they are available from PubMed, they should be completed. For example, the page number of Ref. 1 and Ref. 2 is 331 and 1180, respectively.

Answer: We would like to thank the reviewer for the elaboration. Changes are made! Page numbers are added.