PONE-D-21-35714Identification of avian hepevirus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in PolandPLOS ONE

Dear Dr. Matczuk,

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: SUMMARY OF THE MAIN FINDINGS

In this manuscript, the authors presented the findings of a study designed to investigate the prevalence of avian hepatitis E virus (aHEV) in chicken flocks (broilers, laying hens and broiler breeders), ducks, geese and turkeys aged 1-60 weeks from 307 flocks located in different parts of Poland. Some captive Western capercaillies (Tetrao urogallus) were also included in the study. Using reverse transcription of viral RNA isolated from liver and spleen samples and nested polymerase chain reaction, the authors detected aHEV genetic material in 34 (10.1%) of 336 samples. The highest aHEV infection rate was obtained in broiler breeder flocks (14/40, 35.0%) followed by laying hens (9/49, 18.4%), broilers (7/53, 13.2%), Pekin ducks (2/35, 5.7%) and geese (1/99, 1.0%). Only one (3.5%) of 29 Western capercaillies samples was positive while no viral RNA was detected in the turkey flocks. Phylogenetic analysis based on nucleotide sequences of the partial helicase gene showed that 22 of the identified sequences belonged to genotype 2, 11 to genotype 4, and one to genotype 3 with the genotype 2 sequences forming three distinct clusters. Authors concluded that this is the first detection of aHEV genotype 2 in domestic geese and ducks, and first detection of aHEV genotype 4 in Poland. In addition, they noted that aHEV is most prevalent in broiler breeder flocks in Poland and demonstrated its presence among Western capercaillies, indicating that this avian species is susceptible to the virus.

I believe the manuscript has sufficient quality for publication in PLOS ONE provided the authors can adequately address all the specific comments/issues raised below.

DETAILED REVIEW REPORT

General comments

In my opinion, this manuscript is well written and technically sound. The contents fall within the scope of PLOS ONE and reflect original contribution to knowledge in the field. The methods have been described in sufficient details to allow for reproducibility, while the interpretations are consistent with the objectives of the study and conclusions are justified by the data.

The organization of the article is generally satisfactory although there are some areas where the authors seemed to assume that the reader knows the information they are trying to pass across. For example, the specific locations of the sampling sites in Poland were not given (Lines 75-76), the type of “staff” being referred to in Line 199 was not specified, and what the farms should be “closely monitored” for as well as the host species in which aHEV pathogenicity study should be conducted were not mentioned (Line 239).

In addition, there are some areas where the quality of the English Language needs to be improved. It is advisable that authors engage the services of an English Language editor to improve readability of the text.

Specific comments

Title: I suggest the manuscript title be changed to “Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland”.

Abstract

1. Line 24: It is advisable that authors include the total number of samples here so that readers can have an idea of the total population screened by just reading the Abstract e.g. (34/336 samples, 10.1%).

2. Line 25: Same comments as in 1. above apply here.

Introduction

1. Lines 38, 44, 48 and 233: Authors should endeavor not to start a sentence/paragraph with aHEV. Rather, they should render it as “Avian HEV”.

2. Lines 64-68: Authors have presented results of the study in this introductory section. These should be relocated to the appropriate section in the manuscript.

Materials and methods

1. Lines 71-72: (a) It is important that authors state the voivodeships and counties of Poland from where these samples were collected.

(b) Were these samples from sick or apparently healthy birds? Please specify.

2. Lines 73-74: This period of sample collection is different from the duration stipulated in the Abstract (Lines 19-20). Please reconcile the two.

3. Lines 75-76: The locations of these poultry facilities in Poland should be stated. The map of Poland shown in Figure 2 should have the names of the voivodeships or counties from which samples were collected and those with positive samples.

4. Line 76: Authors should be specific about the internal organ samples being referred to here.

5. Lines 76-77: The age(s) of the Western capercaillies should be stated here.

6. Line 80: Please give examples of these diagnostic procedures to which the samples were subjected.

7. Lines 85-86: Were the Western capercaillies samples from dead or live birds? Please give more information on these birds and how the 29 samples from them were collected.

8. Line 88: Authors should mention the tissues from which viral RNA was extracted.

9. Line 93: Why "first" nested PCR? Did you perform two nested PCR procedures?

Results

1. Line 114: I suggest that authors provide a breakdown of these 34 positive samples into liver and spleen. This might give an indication of the sample that is better suited for aHEV detection.

2. Lines 123-126: The implications of these findings should be highlighted in the Discussion section, otherwise they add no value to the work and should be deleted.

3. Lines 151-152: Supplementary Table S1 is not clear at all. So, it could not be used to confirm the percentage nucleotide identities being referred to in Lines 146-151.

4. Line 159: It would be nice for authors to give the names of locations from where the positive samples were obtained in this map since not all readers are familiar with the different voivodeships and counties in Poland.

Discussion

1. Line 169: I suggest authors retain the description that has been used up until now i.e., aHEV infection instead of BLS. So, this should be changed to "the overall prevalence of aHEV".

2. Line 174: Authors should be consistent with the description of the virus throughout the write-up. You should either retain the use of aHEV or Orthohepevirus B rather than using the two interchangeably.

3. Lines 176-178: I suggest authors reference in their Discussion recently published work such as Osamudiamen et al. (2021) who detected aHEV RNA in tested serum and fecal samples from layer chickens of various ages in Nigeria.

4. Line 206: In my opinion, the sentence will read better if it begins with "Most of the analyzed sequences"

5. Line 217: Authors mentioned in the Results (Line 150-151) that 11 sequences belonged to genotype 4. Is it all the 11 that were 82.2% identical to the Hungarian aHEV sequence or just one of them? Please clarify.

Line 220: I suggest that the sentence starting from “Another” should be the beginning of another paragraph.

Generally, I observed that the Discussion contains too many short paragraphs which reduces the quality of the manuscript. It is advisable that authors merge paragraphs with similar content so that the Discussion can be compact and have a good flow to it. For example, the paragraph that starts on Line 205 should be merged with the one that starts on Line 209.

Lines 227-229: FAdv-1 is a virus and cannot have "virucidal effect". Rather, it is calcium hydroxide and glutaraldehyde that have such effect. So, the sentence should be recast.

Line 231: Replace “virucidals” be with "virucidal agents".

Line 232: I suggest that authors include some few lines on their findings on Western capercaillies in this conclusion section.

Reviewer #2: Matczuk and colleagues present an interesting study on the prevalence of aHEV RNA in different bird species in Poland. The colleagues analysed 336 samples from farmed commercial layer hens, breeder broilers, broilers, ducks, geese, turkeys and also from western capercaillies from Poland’s State Forest Districts.

As a result, the authors detected aHEV RNA in 10.1% of the samples with the highest prevalence in broiler breeders (35%), while the prevalence was low in ducks (5.7%) and geese (1%) and no RNA was detected in turkeys. Also, 1 of 29 samples from western capercaillies was aHEV RNA positive. Most samples belonged to aHEV genotype 2 according to previous studies. For the first time, aHEV genotype 4 has been detected in Poland.

Overall, the study presents new interesting data, shedding more light on the diversity of circulating aHEV strains in Poland. However, there is conflicting information concerning the terms samples/flocks as well as testing strategy (pooled vs. individual testing) raising questions on the resulting prevalence data. Further, phylogenetic analysis based on the Neighbor-joining method is rather outdated and should be performed using the Maximum-likelihood method for a more reliable outcome.

Specific comments

Introduction:

The authors should include the recent reports about potential novel aHEV genotypes and aHEV from wild birds (e.g. Su etal 2018 DOI: 10.1111/tbed.12987 ; Reuter etal 2016 DOI: 10.1016/j.meegid.2016.10.026; Osamudiamen etal 2021 DOI: 10.3390/v13060954).

Lines 63-43: The authors should clarify that the included species were not tested before in Poland, as duck and geese e.g. were tested positive for aHEV in China.

Materials and Methods:

Lines 73-74: In this section, the study was carried out between 2019-2020 while in the abstract it is 2018-2019 (see Abstract, lines 19-20). Please correct.

Lines 73-74: Is there any information on the health status of the birds before sample collection?

Line 74: Were liver and spleen samples pooled? If not, was there any difference in the proportion of positive samples between these 2 tissues?

Line 76: Which internal organ samples? Please give more details.

Line 91: Which primer? Please give the sequence.

Line 101: Were the products sequenced in both directions? Please give this information.

Results:

Line 114: According to the Material and Methods section, the samples were pooled and not tested individually, so how can it be 34 out 336 samples? Or were sample in positive tested pools re-tested individually? Please clarify. Also, “sample” seems to be used synonymous with “flock”. This indicates that each flock is represented by only one sample as nSample = nFlocks in the Materials and Methods section. Please describe more clearly what is meant with “sample” and “flock” and give the number of each collected and tested positive in the Material and Methods and the Results section in a clear way.

Line 117: It must be “18.4%” instead of “18,.%”

Table 1: The average is not correct for all sample types (Laying hens and broilers). Please re-calculate.

Lines 123-126: For broilers the average age of infected vs. uninfected birds is the same. Further, are the results significant? A statistical analysis should be performed.

Figure 1: Please indicate in the legend how long the sequences used for phylogenetic analysis were, as probably the primer sequences were trimmed before analysis?

Figure 1: Sequences of putative novel genotypes which were recently detected in chickens should be included in the phylogenetic analysis. Also, a maximum-likelihood tree is better for phylogenetic analysis as the results are more reliable.

Figure 1: The animal species could be indicated for the sequences from the previously performed study.

Figure 1: For better visibility, the sequences from this study should be marked in bold or otherwise.

Line 146: Please specify which “sequence identity”; nucleotide or amino acid?

Discussion:

Line 169: The chickens are aHEV RNA positive. This does not automatically mean that they have BLS. Or did they show signs of disease?

Lines 173-178: There are also aHEV RNA prevalence data from e.g. Spain or Nigeria (Peralta etal 2009 DOI: 10.1016/j.vetmic.2008.12.010 ; Osamudiamen etal 2021 DOI: 10.3390/v13060954) which should be discussed as it is not clear why for comparison only data from a Chinese study have been chosen. Also, the authors could include their own RNA prevalence data from their previously performed study.

Lines 203-204: It could also mean that the primers which were used are not able to detect aHEV from turkey. Please include this.

Lines 209-210: The first outbreak of what? Please specify.

Line 222: Table S2 is meant.

Conclusion:

Line 233-234: The first sentence seems to be incomplete. aHEV is the most “what” in Poland? Do you mean that aHEV is highly prevalent?

Table S2: The sequences from this study should rather be named by the isolates name and the accession number maybe in brackets to simplify the comparison of the host species of the old and new samples.

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Reviewer #1: No

Reviewer #2: No

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Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland

PONE-D-21-35714R1

Dear Dr. Matczuk,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Yury E Khudyakov, PhD

Academic Editor

PLOS ONE

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