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

Orthohepevirus B, commonly known as avian hepatitis E virus (aHEV), causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS) in chickens. BLS is an emerging disease among chicken flocks in several countries around the world. In our previous studies, serology and molecular biology screening revealed that chicken flocks are widely affected by aHEV in Poland. The present study, which was conducted between 2019 and 2020, aimed to investigate the prevalence of aHEV in chicken flocks and other poultry, including ducks, geese, and turkeys. A total of 307 flocks were examined. In addition, 29 samples from captive wild birds (western capercaillies, Tetrao urogallus) were analyzed. In all the investigated poultry species, except turkeys, the nucleic acid sequence covering part of the ORF1 gene of the aHEV genome was detected (34/336 samples, 10.1%). The infection rate was found to be the highest in broiler breeder chicken flocks (14/40 samples; 35%). Phylogenetic analysis of partial ORF1 gene, which encodes helicase, revealed that the obtained sequences belonged to genotypes 2 and 4, while one belonged to genotype 3. Genotype 2 was detected for the first time in domestic geese and ducks, and genotype 4 was detected for the first time in Poland. The study demonstrated the presence of aHEV among the investigated western capercaillies, suggesting that this species is susceptible to aHEV infections and biosecurity is therefore required in western capercaillie breeding facilities.

Introduction

Orthohepevirus B, which is also known as avian hepatitis E virus (aHEV), belongs to the Hepeviridae family within the genus Orthohepevirus. Four species of Orthohepevirus have been identified so far (Orthohepevirus A, B, C, D). Among them, only Orthohepevirus B species include HEV isolates from chickens and wild birds, while the other three species comprise HEV isolates from mammals [1].

Avian HEV is a nonenveloped, single-stranded RNA virus. Its genome has a size of 6.6 kbp and consists of three open reading frames (ORFs) and nonencoding regions at 5’- and 3’-end [2]. ORF1 encodes the domains for nonstructural polyproteins, such as RNA helicase, which are involved in replication. The viral capsid protein (ORF2)-encoding gene is situated at the 3’-end of the genome, overlapped by ORF3 encoding a cytoskeleton-associated phosphoprotein [2-4].

Avian HEV causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS). It is most prevalent among breeder broiler flocks and commercial laying hens. The disease was observed for the first time in the 1980s among chickens in Australia, and later it was reported in the 1990s in North America [3, 5]. In Europe, it was first described in 2004 in Italy, while in Poland it was detected in 2010 [6, 7]. Avian HEV was identified as an etiological agent of HSS and BLS in the years 1999 and 2001, respectively [3, 8]. The pathogenesis of aHEV infections is still unknown because the virus cannot be efficiently propagated in cell culture and replicates only in infected animals [4].

Thus far, four genotypes of Orthohepevirus B have been proposed, but the International Committee on Taxonomy of Viruses ICTV is yet to recognize them. The proposed genotypes share approximately 80% nucleotide identity [7, 9]. In Europe, genotypes 2, 3, and 4 have been observed among chicken flocks, while genotype 1 was detected only once from a sample obtained from feral pigeons [10-13]. Additionally to domestic birds, a novel orthohepevirus distantly related to aHEV, was sequenced from wild birds little egret sampled in Hungary [14].

BLS-affected chickens typically present with enlarged livers and spleens and blood-stained fluid in the abdomen. In addition, a significant decrease in egg production (10%–40%) and an elevated mortality rate (1%–4%) were reported [15]. Since 2016, a new disease associated with aHEV infection has been noticed in layer and broiler breeder hens in several provinces in China. Infection caused hepatic rupture hemorrhage syndrome and was associated with high mortality in hens [16].

Both anti-aHEV antibodies and viral RNA were detected in healthy chicken flocks. This indicates that aHEV is capable of causing subclinical infections [11, 17]. Recent study from apparently healthy layer flocks from Nigeria indicate approximately 10% aHEV RNA prevalence, while 75% of the examined flocks show antibodies against aHEV [18, 19]. This study assessed the prevalence of aHEV among Polish flocks. The analyzed bird species included those that were not previously tested in Poland such as geese, ducks, turkeys, and western capercaillies.

Materials and methods

Sample collection

For genetic identification of aHEV isolates, liver and spleen samples were collected from different poultry species: laying hens (n = 49), breeder broilers (n = 40), broilers (n = 53), ducks (n = 35), geese (n = 99), and turkeys (n = 31). Sample collection was carried out between 2019 and 2020. The sample obtained from one flock consisted of livers and spleens collected from five birds (pooled sample). The samples originated from flocks with difficult rearing and increased deaths. In the case of four flocks, veterinary practitioners requested a PCR test for aHEV presence, these flocks were indicated in Table 1.

Table 1

Description of positive sequences obtained in this study.

No.	Isolate	GenBank accession number	Species	Age (weeks)	Year of isolation	Industry-farm	Genotype/cluster
1	1/CL/2020/PL	OK423501	Laying hen	40	2020	1-A	4
2	2/CL/2020/PL	OK423502	Laying hen	35	2020	2	2/1
3	3/CL/2020/PL	OK423503	Laying hen	55	2020	2	2/1
4	4/CL/2020/PL	OK423504	Laying hen	50	2020	1-B	2/3
5	5/CL/2020/PL	OK423505	Laying hen	24	2020	19	2/1
6	1/BB/2020/PL	OK423506	Breeder broiler	31	2020	3	2/1
7	2/BB/2020/PL	OK423507	Breeder broiler	28	2020	4	2/1
8	3/BB/2020/PL	OK423508	Breeder broiler	44	2020	5-A	2/3
9	4/BB/2020/PL *	OK423509	Breeder broiler	44	2020	5-A	2/3
10	5/BB/2020/PL	OK423510	Breeder broiler	45	2020	6	2/2
11	6/BB/2020/PL	OK423511	Breeder broiler	40	2020	7	2/2
12	7/BB/2020/PL	OK423512	Breeder broiler	60	2020	7	2/3
13	8/BB/2020/PL	OK423513	Breeder broiler	30	2020	8	2/3
14	9/BB/2020/PL	OK423514	Breeder broiler	48	2020	6	2/2
15	10/BB/2020/PL	OK423515	Breeder broiler	12	2020	9	2/1
16	11/BB/2020/PL	OK423516	Breeder broiler	77	2020	10	2/3
17	12/BB/2020/PL	OK423517	Breeder broiler	42	2020	11	3
18	13/BB/2020/PL	OK423518	Breeder broiler	48	2020	1-G	2/1
19	1/B/2020/PL	OK423519	Broiler	2	2020	12	4
20	2/B/2020/PL	OK423520	Broiler	3	2020	12	4
21	3/B/2020/PL	OK423521	Broiler	4	2020	13	4
22	4/B/2020/PL	OK423522	Broiler	5	2020	12	4
23	5/B/2020/PL	OK423523	Broiler	3	2020	14	4
24	6/B/2020/PL	OK423524	Broiler	3	2020	12	4
25	1/D/2020/PL	OK423525	Duck	6	2020	15	4
26	1/G/2020/PL	OK423526	Goose	2	2020	16	2/3
27	1/CL/2019/PL *	OK423527	Laying hen	40	2019	1-D	2/3
28	2/CL/2019/PL *	OK423528	Laying hen	37	2019	1-F	4
29	3/CL/2019/PL *	OK423529	Laying hen	28	2019	1-C	2/3
30	4/CL/2019/PL	OK423530	Laying hen	30	2019	1-E	4
31	1/BB/2019/PL	OK423531	Breeder broiler	35	2019	5-B	2/3
32	1/B/2019/PL	OK423532	Broiler	6	2019	17	4
33	1/D/2019/PL	OK423533	Duck	2	2019	18	2/3
34	1/WC/2018/PL	OK423534	Western capercaillie	80	2018	Poland’s State Forest	2/2

* Samples obtained from flocks directed to the aHEV PCR test by veterinary practicioners.

The study included a total of 307 flocks, which were aged 1–77 weeks and procured from different poultry facilities located in Poland. The study covered 11 polish voivodeships: Pomeranian, Lubusz, Greater Poland, Lower Silesia, Masovian, Łódź, Opole, Silesian, Lesser Poland, Lublin and Subcarpathian. Between 2018 and 2019 dead western capercaillies (n = 29) from Poland’s State Forest Districts were sent for necropsy and sample collection. The age of these birds was between 1 to 80 weeks. In this case, liver and spleen were also pooled for RNA extraction, but the one sample was taken from each bird.

The collected samples were stored at –80°C for further analysis.

Ethical approval

The study was conducted on clinical samples, either dead birds, or internal organ samples collected during on-site necropsies, sent by veterinary practitioners. The use of such samples does not require approval from the Local Ethical Committee, according to Polish animal experiment regulations.

Isolation and reverse transcription of viral RNA

Viral RNA from pooled livers and spleens was extracted using a Total RNA Mini Plus Kit (A&A Biotechnology, Gdynia, Poland). Using the extracted RNA, cDNA was synthesized with a Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Poland), as per the manufacturer’s instructions. An external reverse primer Helic R-1 (5′-CCTCRTGGACCGTWATCGACCC-3′) was used for cDNA synthesis [13, 17].

Amplification of partial helicase gene

For the screening of aHEV-positive samples, the nested polymerase chain reaction (PCR) assay was performed with two degenerate primers sets that targeted the partial helicase gene region in ORF1, as described previously [13, 17]. A previously sequenced aHEV fragment was used as a positive control (GenBank accession number MH636899.1). The size of the obtained PCR product was 386 bp.

The products resulting from second-round PCR were examined on a 2% agarose gel stained with Midori Green DNA Stain (Nippon Genetics Europe GmbH, Düeren, Germany). The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.

Sequence analysis

The resulting sequences were compared with that of aHEV reference strains from established genotypes and sequences previously published in Poland, using the BLASTn alignment algorithm at the GenBank database (NCBI). Then, the sequences were aligned using ClustalW and Sequence Identity and Similarity (SIAS) online tool [20].

Phylogenetic trees were constructed using the Maximum-likelihood method and the Neighbor-Joining method with 1000 bootstrap replicates and Kimura 2-parameter model using MEGA 7.0 software [21].

The acquired sequences were deposited in the GenBank database under accession numbers OK423501 to OK423534.

Statistical analysis

Statistical analysis was performed with Prism 9 (GraphPad, USA). Statistical analysis was performed with unpaired t-test with Welch’s correction. Statistical analysis of age of geese samples were performed with unpaired t-test. Minimal data set is contained in S1 Data.

Results

The aHEV genetic material was detected in 34 out of 336 tested samples (10.1%). Infection was found to be the most prevalent in broiler breeder flocks, and detected in 14 out of 40 tested flocks (35%). In commercial laying hens, the aHEV RNA was detected in 9 out of 49 flocks (18.4%), while in chicken broilers, Pekin ducks, and geese flocks there were 7 positives out of 53 (13.2%), 2 out of 35 (5.7%), and 1 out of 99 tested flocks (1%), respectively. One sample was positive from 29 tested western capercaillies from the same captive flock. No viral RNA was detected in the tested turkey flocks. The obtained results, including flock age upon testing, are presented in Table 1.

There were no statistical differences between the average age of aHEV positive flocks and the average-aged tested birds when each production type was analyzed (Table 2).

Table 2

Average age of infected flocks versus average age of examined flocks.

	Average age of infected flocks (weeks)	Average age of examined flocks (weeks)	P-value	Significance
Commercial layer	37.7	31.1	0.12	ns
Breeder broiler	41.7	35.8	0.23	ns
Broiler	3.7	4.1	0.50	ns
Duck	4.0	5.0	0.7	ns
Goose	2.0	5.7	0.38	ns
Turkey	-	6.2	-	-

Statisticasl analysis was performed with unpaired t-test with Welch’s correction (ns—not significant).

Phylogenetic analysis based on the partial helicase gene showed that most of the sequences belonged to genotype 2 (n = 22), 11 belonged to putative genotype 4, and only 1 belonged to genotype 3. In the Neighbor-Joining phylogenetic tree, the sequences of genotype 2 formed three separate clusters (Fig 1). Genotype 2 was found to be dominant in all the tested species. However, in broilers, only genotype 4 was identified. Genotype 3 was detected in one of the broiler breeder flocks, while genotype 4 was detected in three flocks of commercial layers and one flock of ducks. The geographical distribution of these sequences with regard to phylogenetic clusters and poultry species is shown in (Fig 2).

Fig 1

Phylogenetic analysis based on the partial nucleotide sequences of helicase gene.

Similar sequences and reference genotype sequences were acquired from GenBank database. Sequences obtained in this study are marked with a red line and described in Table 1. The phylogenetic tree was constructed using the Neighbor-Joining algorithm with 1000 bootstrap replicates. Bootstrap values are shown on the tree. CL—laying hen, BB—broiler breeder, B—broiler, D—duck, G—goose, WC—western capercaillie. Symbols: stars, squares, circles and triangles indicate the same farm source of sequence.

Fig 2

Geographical distribution and genotype classification of avian HEV viruses.

(A) The location of voivodeships together with the number of infected flocks/number of tested flocks. (B) Poland is divided into voivodeships and counties. Farms belonging to the same industry are marked with a triangle or a circle symbol. Numbers in parentheses are referred to genotype 2 clusters. Commercial laying hen farms marked in black, breeder broiler farms in green, broiler farms in red, duck farms in blue, goose farms in orange and western capercaillies breeding facility in purple. Map reprinted and edited from [22] under a CC BY license, with permission from Wikimedia Commons, original copyright [2020].

An analysis of the sequences obtained in this study revealed a nucleotide sequence identity of 79.35%–100%. Whereas nucleotide sequence identity was between 74,63% and 96,46% when compared to sequences from GenBank (excluding Polish sequences). All the details are provided in the percent identity matrix in S1 Table.

Discussion

Poultry production is constantly and rapidly expanding in Poland. The country has been the leading producer of poultry meat in the European Union since 2014. After the detection of BLS, there has been an increased awareness in Poland about the diseases that can impede reproduction and are capable of causing mortality and affecting egg laying performance.

In this study, the overall prevalence of aHEV was found to be 10.1% in the analyzed poultry production types and animal species. However, more significant differences in the prevalence of aHEV viral RNA was noted in the broiler breeder flocks (35% flocks tested), followed by commercial laying hens (18.4%) and broiler flocks (13.2%). A recent Chinese study from the years 2018–2019 showed that the prevalence of aHEV RNA in chickens was 7.92%. In the cited study, only samples from the flocks of age 17–20 weeks were tested. In some Chinese provinces, the prevalence was higher (e.g., 13.85% in Hebei province) [23]. The results on aHEV prevalence might be influenced depending on the type of sample tested. In 2009 study from Spain, where random serum samples were tested for the presence of aHEV RNA, only 1,66% of samples were positive. When fecal and serum material from healthy 29 layer and breeder flocks were tested, 3,45% of samples were aHEV RNA positive [11]. Recent study performed on apparently healthy layer flocks from Nigeria indicates approximately 10% aHEV RNA prevalence. In this study the prevalence in layer chicken flocks was 18,4%, which is higher, then prevalence rates in Nigeria [19]. This could be simply due to the type of material studied, as in Nigeria apparently healthy chickens were examined, while in our study we had samples obtained from flocks with diminished performance. In a recent study, we reported that the seroprevalence of aHEV in chicken flocks was much higher in Poland (56.1%) compared to China (35.9%), whereas in southwest Nigeria 75% examined chicken flocks were positive for aHEV antibodies [13, 18, 24].

The present study indicated that the infection rate in Pekin ducks and geese was minimal, suggesting that these birds are not the main reservoir of aHEV. Data available on the aHEV infection of domestic waterfowl in the literature are limited. Seroprevalence and molecular biology studies have been performed only in chicken flocks and wild birds [4]. In one study, ducks and geese were shown to be infected with aHEV genotype 3. The tested birds were housed in a mixed-type farm along with infected chickens [25].

The occurrence of aHEV has also been demonstrated in free-living birds such as little egret, song thrush, little owl, feral pigeon, and common buzzard [12, 14]. So far, aHEV viral RNA has not been detected in wild Anseriformes. Similar to chickens and turkeys, western capercaillies belong to the family of Phasianidae. These birds are protected species in Poland and are bred in three facilities in the country. Some of them are brought back to the forests as part of the reintroduction program. In this study, aHEV viral RNA was detected in one only bird. The sequence belonged to genotype 2 and was very similar to the nucleotide sequence obtained from broiler breeder 6/BB/2020/PL (99.1% identity) and those detected in broiler breeder flocks in Poland in the years 2017 and 2018. The prevalence of aHEV in wild birds has not been investigated in Poland. Few studies available from other countries have demonstrated that aHEV detected in domestic poultry and wild birds are distinct lineages [12, 14]. A high similarity between the sequence obtained from western capercaillie and the sequences from broiler breeder chicken is suggestive of indirect transmission, by contaminated feed or water, or caretaker or veterinary staff, and not direct transmission from wild birds.

In this study, aHEV infection was not observed in turkeys. Turkeys were experimentally infected with aHEV [26], but the viral RNA has not been isolated from these species under field conditions. The lack of infection in turkeys can be explained by the fact that the highest bioasecuration standards are followed in turkey farms. Another explanation is that the primers used in this study are not able to detect aHEV circulating in turkey population.

This study involved an analysis of the helicase gene fragment that led to the identification of three different genotypes. Most of the analyzed sequences belonged to genotypes 2 and 4, while only one belonged to genotype 3, which indicates the high genetic diversity of aHEV in Poland. Genotype 3 was detected previously in chickens in Poland during the first aHEV outbreaks [7]. In the present study, only one sequence was found to belong to genotype 3, which shows that this genotype is still present in the field, but not dominant in Poland. This sequence had 96.5% identity to the one detected in Canada in 2003. In a recent study conducted by our group in Poland in 2017–2018, genotype 2 was observed to be the most prevalent [13]. Phylogenetic tree based on partial helicase gene showed that the gene sequences were divided into three clusters, as can be seen in the phylogenetic tree presented in this work (Fig 1). Genotype 4 is described here for the first time in Poland. The sequences belonging to this genotype was 82.0% - 86,7% similar to the sequence reported from Hungary in 2010 which was detected in only one liver sample from BLS outbreaks [27]. Interestingly, this genotype was predominant in one voivodeship (Greater Poland), not in broiler breeder farms but in duck flocks, broiler flocks, and commercial layer flocks.

It is still unknown, whether infections with aHEV are age-associated or not. Conflicting results were described so far and are reviewed in [4]. In our study, there was no statistically significant difference between the age of the tested and infected birds in individual species or chicken production types. We could not statistically analyse all positive versus tested samples, because of the age bias of the flocks from where samples were taken. We had small amounts of samples from young commercial layer flocks or broiler breeder flocks, or samples from older ducks or geese.

In this study we classified genotypes according to analysis performed with the Neighbor-Joining method, because the vast majority of previous publications on aHEV genotyping were performed with this method. However, when the Maximum-likelihood method was used the cluster 3 of genotype 2, seems to be a distinct genotype (S1 Fig.). It should be reminded that thus far, the ICTV does not recognize genotypes of Orthohepevirus B. With the emergence of new sequences and genotypes it is urgent to provide proper description of aHEV genotype and appropriate method to distinguish them.

Another interesting observation is that some samples were from the same farms from where we obtained samples and detected sequences in our previous study (Fig 1, S2 Table) [13]. Similar sequences of aHEV partial ORF1 gene were detected from samples collected from those farms, with an exception of the farms indicated with a black square, where sequences were found to belong to genotype 2 in 2017, and to genotype 4 in 2020. This implies that certain aHEV are present on the same farms, which indicates their persistence. Indeed, enteric nonenveloped viruses can resist the action of disinfection agents. Recent data on the virucidal agents effect on nonenveloped fowl aviadenovirus 1 (FAdV-1), which causes gizzard erosion in chickens, suggest that calcium hydroxide and glutaraldehyde can act as effective disinfection agents at an adequate concentration and ambient temperature [28]. However, there are no data regarding effective virucidal agents against aHEV in field conditions.

Conclusions

The highest prevalence of aHEV was observed in broiler breeder flocks in Poland. Of the four genotypes of aHEV, genotype 2 has been shown to be widespread so far. Genotype 4, which is newly detected and observed for the first time in Poland, should be investigated in detail with respect to its pathogenic potential in chickens. Natural aHEV infection can be possible in farm ducks, geese and western capercaillies held in captivity. Phylogenetic analysis suggests, that the source of infection is most likely related to indirect transmission from chicken farms. Based on this study, further research should be conducted on aHEV pathogenicity in species other than chickens.

Phylogenetic tree based on maximum-likelihood method.

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Percent identity matrix of the nucleotides for partial ORF1 among avian HEV strains included in phylogenetic analysis.

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aHEV isolates collected in the same region in this and previous study from 2017–2018.

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Minimal data set for statistical analysis performed in Table 2.

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12 Jan 2022

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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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5 Apr 2022

Dear Reviewers,

The co-authors and I would like to thank you for your precious time as well as for your valuable and kind comments considering our manuscript and further revisions required.

We have been able to incorporate changes to reflect the suggestions provided by the reviewers. All amendments in the manuscript are highlighted. The format of the manuscript has been revised according to the submission guidelines.

Here is a point-by-point response to the reviewers’ comments and concerns:

Response to comments and suggestions of Reviewer#1

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.

Answer (Ans): We would like to thank you again for the review. We have revised our manuscript according to the Reviewer’s comments and suggestions (please find all changes within the manuscript highlighted in red font. The orginal manuscript was edited by English Langueage editor, we added the file the certificate of the analysis.

Lines 75, 199, and 239 were corrected. The locations have been specified in the text of the manuscript and marked in the corrected Fig 2.

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”.

Ans: We are thankful for this suggestion. The title has been changed according to the Reviewer’s suggestion.

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.

Ans: In the lines 24 and 25 corrections have been made as suggested.

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”.

Ans: Thank you for the suggestion, the sentences have been corrected.

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.

Ans: This paragraph has been removed to not repeat the information about results in the introduction section.

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.

Ans: Additional information about the voivodeships of Poland from where these samples were collected, was added in the text. While the counties were marked on the edited map to make the manuscript more clear. We hope that the Reviewer will agree with this solution.

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

Ans: The answer to the Reviewer’s question is included in the modified sentence:

“The samples originated from flocks with difficult rearing and increased deaths. In the case of four flocks, veterinary practitioners requested a PCR test for aHEV presence, these flocks were indicated in Table 1.”

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.

Ans: We apologize for this oversight. The amendment was introduced in the text.

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.

Ans: We agree with this comment. Therefore, we added the locations of poultry flocks and edited the map.

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

Ans: Liver and spleen samples were taken from western caperacillies too. This sentence has been modified according to the Reviewer's recommendations.

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

Ans: Thank you very much for this comment. We provided information about the age of these birds.

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

Ans: We thank the Reviewer for pointing this out. The phrase "and subjected them to different diagnostic analyses" has been removed from this sentence

The answer to reviewer question is:

All of the samples were subjected to standard bacteriological examination. Some flocks were subjected to PCR tests to detect: IBD infectious bursal disease virus , MDV Marek’s disease virus, FadV-1 fowl adenovirus 1, Reovirus. 4 flocks were specifically subjected for aHEV PCR detection. They were labelled in table 1. The duck and geese flocks were subjected to qPCR against GPV goose parvovirus that detect classical GPV and variant nGPV, GHPV goose hemorrhagic polyoma virus, some flocks were subjected to PCR for Mycoplasma sp. detection. Western capercilles samples were subjected to bacteriological examination.

However we think that supplementing this information would have no impact on the scientific quality of the manuscript. We hope that the Reviewer will agree with this statement. However, in Table 1 we marked the sequences from flocks that were specifically sent for aHEV PCR.

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.

Ans: We checked again and dead western capercaillies (whole birds) were sent for the necropsy. These necropsies were performed by co-author prof. Alina Wieliczko who collected material for storage.

To accomodate also comments from Reviewer 2, we modified the paragraph accordingly.

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

Ans: This missing information was added.

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

Ans: The word "first" has been used unnecessarily. We performed one nested PCR procedure.

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.

Ans: We thank the Reviewer for this suggestion.

In fact in our previous paper doi: 10.1007/s00705-018-4089-y we analysed RNA positivity in spleen and liver samples.

To sum up that study aHEV was detected twice as often in liver then in spleens. There were some birds 2/18 that had detectable aHEV RNA only in spleen and not in liver. Taking it into account we decided to pool liver and spleen samples.

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.

Ans: With suggestion from reviewer 2 we made statistical analysis on this data. There is no statistical difference, Table 2 was changed, and this result was discussed, lines 387-393 of R1 manuscript.

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.

Ans: S1 table resolution was checked. We think that for data availability it is good to have this table included in the supporting information.

The result on similarity was from BLASTn and it did not cover all the sequences. The result was changed and now reflects the data presented in S1 Table.

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.

Ans: The exact location cannot be given as this is sensitive data. The location of the farm is just to see if there are geographical clusters of positive flocks. Names of voivodeships were added to the Fig. 1 map. Also the figure now contains the information about all the samples tested. We could not put all the negative flocks on the map as it would be unreadable, but we included numbers of samples tested for each voivodeships. We hope that these changes add value to the manuscript.

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.

Ans: On the indicated lines 169 and 174, corrections have been made according to the Reviewer’s suggestion. We decided to retain aHEV since it is better known and simpler name then Orthohepevirus B.

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.

Ans: We are thankful for this hint. We agree with this and have incorporated your suggestion.

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

Ans: Thank you for the suggestion, the sentence has been corrected.

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.

Ans: Indeed, 82,2 % is the value for just one sequence. The sentence was corrected, and values for every sequence from that cluster was added, which was: 82.5% - 86,7%. Additionally, we added also reference sequences from genbank to supplementary table S1. Therefore one can check the similarity of each sequence.

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

Ans: In accordance with the recommendations another paragraph was added.

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.

Ans: Mentioned paragraphs were merged. Additionally two more paragraphs were merged. There are 2 new paragraphs of discussion regarding phylogenetic method and age of the birds.

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.

Ans: Thank you for noticing this mistake. The sentence has been corrected.

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

Ans: The word „virucidals” was replaced in the manuscript with „virucidal agents”.

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

Ans: Information on findings on western capercaillies was added to conclusion section.

Response to comments and suggestions of 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.

Answer (Ans): We would like to thank you again for the review. We have revised our manuscript according to the Reviewer’s comments and suggestions (please find all changes within the manuscript highlighted in green font.

General comments: The types of samples (pooled for the poultry and individual for the western capecraillies) as weel as pooling strategy liver+spleens were explained in more detail in the manuscript’s materials and method section. We hope that this section is clearer now.

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).

Ans: These publications were included in the Introduction section. However, only Reuter et al. describes aHEV from wild birds.

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.

Ans: The sentence was modified; „ in Poland” was added to this sentence.

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.

Ans: We apologize for this oversight. The sentence has been corrected.

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

Ans: We are thankful for this suggestion. All the samples were from flocks with difficulties with production or elevated death rates. This information was included in the manuscript lines: 85-87.

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

Ans: In fact in our previous paper doi: 10.1007/s00705-018-4089-y we analysed RNA positivity in spleen and liver samples.

To sum up that study aHEV RNA was detected twice as often in liver then in spleens. There were some birds 2/18 that had detectable aHEV RNA only in spleen and not in liver. Taking it into account we decided to pool liver and spleen samples.

Therefore in this study liver and spleen samples were pooled. Additional sentence with rationale of the sampling processing was added.

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

Ans: Liver and spleen samples were taken from western caperacillies too. This sentence has been modified according to the Reviewer's recommendations.

Line 91: Which primer? Please give the sequence.

Ans: Thank you for pointing out the missing primer sequence, it has been added to the manuscript.

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

Ans: The answer to the Reviewer’s question is included in the below sentence: “The amplified products of ORF1 were excised, purified using Gel-Out (A&A Biotechnology, Gdynia, Poland), and directly sequenced in both directions with Sanger’s method (Eurofins Genomics Sequencing GmbH, Cologne, Germany) with the use of PCR primers.”

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.

Ans: Indeed the description was misleading due to different naming of farm animal samples versus samples from western capercailles.

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

Ans: Thank you for pointing this out. Correction has been made.

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

Ans: Table 1 and 2 were corrected. There were some mistakes regarding age of the birds that were corrected.

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.

Ans: We checked the table again and indeed there was a mistake. The table was corrected. The statistical analysis was performed and is described in the method section and in table legend.

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?

Ans: 339 nucleotide long fragment was analysed. Information was added to the figure.

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.

Ans: Only two additional sequences were included in the phylogenetic tree that had a complete genome accessible in GenBank. Unfortunately, other recently detected novel genotypes were either characterised by other fragment of ORF1 gene or of the ORF2 fragment. Those fragments do not cover fragments sequenced in this study, therefore cannot be compared here.

Regarding the phylogenetic method used, we agree that most of the recent publication states that maximum likelihood method is better to discriminate differences in viral sequences. However, vast majority of publications describing genotyping of aHEV was performed with neighbour-joining method, which is scientifically sound algorithm. Therefore the genotypes described and placed in GenBank were analysed with NJ method. We performed the maximum-likelihood analysis and obtained tree was put in Supporting figure S3. There is a difference; cluster 3 of genotype 2 seems to be a distinct genotype. The genotyping method for aHEV should be addressed by bioinformatical analysis, but this analysis is beyond scope of this manuscript.

We discuss on it in the discussion section: lines 376-382.

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

Ans: All of the sequences from previous study are from chickens. The same abbreviations to label type of production was added to the figure, e.g. B – broiler.

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

Ans: Sequences from this study were underlined with red. Bold was not possible in our Mega version.

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

Ans: Thank you for this suggestion. We added a missed word “nucleotide”.

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?

Ans: We agree with the Reviewer’s suggestion. It was changed to "the overall prevalence of aHEV".

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.

Ans: We extended the discussion part on prevalence according to the Reviewer’s suggestion.

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

Ans: Thank you for this relevant suggestion. We included this information in the manuscript.

„Another explanation is that the primers used in this study are not able to detect aHEV circulating in turkey population.”

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

Ans: Thank you for pointing this out. The phrase “of avian HEV”has been added.

Line 222: Table S2 is meant.

Ans: The correction has been made according to the Reviewer's suggestion.

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?

Ans: To avoid ambiguities, according to the Reviewer's suggestion, this sentence was modified to: “The highest prevalence of the aHEV was observed in broiler breeder flocks in Poland”.

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.

Ans: The names of the sequences were changed according to the Reviewer's suggestion.

Please let us know if you still have any questions or concerns about the manuscript.

We will be happy to address them.

Sincerely,

Anna Matczuk

Submitted filename: Response to rewievers.docx

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30 May 2022

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,

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Reviewers' comments:

14 Jun 2022

PONE-D-21-35714R1

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

Dear Dr. Matczuk:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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