Surveillance of Human Rotaviruses in Wuhan, China (2019–2022): Whole-Genome Analysis of Emerging DS-1-like G8P[8] Rotavirus

Round 1

Reviewer 1 Report

I reviewed the manuscript entitled “Surveillance of Human Rotavirus in Wuhan China (2019-2022): Whole Genome analysis of Emerging DS-1- Like G8P [8] Rotavirus.  In this study authors conducted a molecular epidemiology study in Wuhan China to evaluate fecal samples from patients from different ages for the presence of Rotavirus type A (RVA). RVA positive samples were characterized by sequencing.

Based on the importance of RVA in the public health, I consider that this manuscript contains valuable information. However, I consider that multiple aspects should be improved in this manuscript.

A)      Introduction should be updated with more information regarding previous studies characterizing RVA in China. Current version looks out of context.

B)      Methodology. Please, provide more information regarding primer sequences used for sanger sequencing. In case of sample collection, provide additional information regarding the inclusion criteria used for this purpose. Provide more details about the external sequences included in this study.

C)      The presentation of the results should be improved. To enrich this section, I suggest providing more information regarding the clinical status of patients included in this study. Was the presence of specific genotypes associated with specific clinical signs? Overall, I suggest authors improving the presentation of the information presented in tables and phylogenetic trees. In table 2, specify what is the meaning of the sequences highlighted in yellow. I think authors should reduce the number of sequences in tables and phylogenetic trees, and just include the most representative ones. All sequences are from China or from other countries. It is confusing in the way that is presented.

D)      Discussion should be drastically improved; it looks very repetitive with the information presented in the results section. Describe with more detail the relevance of this study compared with previous ones conducted in China. What about vaccination? How different is the current vaccine compared with the lineages found in this study. Discuss more about the impact of the results found in this study in the improvement of current public health strategies to control this virus.

Author Response

 Point-by-point response to Reviewer #1

Q1:Introduction should be updated with more information regarding previous studies characterizing RVA in China. Current version looks out of context.

A1: L47-49, updated with more information regarding previous studies characterizing RVA in China.

 

Q2:Methodology. Please, provide more information regarding primer sequences used for sanger sequencing. In case of sample collection, provide additional information regarding the inclusion criteria used for this purpose. Provide more details about the external sequences included in this study.

A2: L294-296 and L301-303, primer sequences used for RT-PCR and sanger sequencing were listed in Table S1; The external sequences included in this study were listed in Table S1 and/or provided the references; L281-282, the criteria were added.;

 

Q3:The presentation of the results should be improved. To enrich this section, I suggest providing more information regarding the clinical status of patients included in this study. Was the presence of specific genotypes associated with specific clinical signs? Overall, I suggest authors improving the presentation of the information presented in tables and phylogenetic trees. In table 2, specify what is the meaning of the sequences highlighted in yellow. I think authors should reduce the number of sequences in tables and phylogenetic trees, and just include the most representative ones. All sequences are from China or from other countries. It is confusing in the way that is presented.

A3: L74-78, the clinical statuses of patients were described briefly. The specific clinical signs were not associated with the specific genotypes of rotavirus in the present study. Patients infected by rotavirus of different genotypes had severe/moderate or mild signs and symptoms;

L114, all strains detected in present study highlighted in yellow;

Table 2, Figure 1-11, the number of sequences in tables and phylogenetic trees were reduced. The representative G8 rotavirus strains with 11 complete CDS genes published in Rotavirus Viral Genomes data base at NCBI (Virus Variation - NCBI (nih.gov)) were chosen for the construction of phylogenetic trees.

 

Q4:Discussion should be drastically improved; it looks very repetitive with the information presented in the results section. Describe with more detail the relevance of this study compared with previous ones conducted in China. What about vaccination? How different is the current vaccine compared with the lineages found in this study. Discuss more about the impact of the results found in this study in the improvement of current public health strategies to control this virus.

A4: L249-260, the repetitive information shown below was deleted (In order to explore the possible origin of the G8P[8] rotavirus detected in Wuhan, the whole genome of simultaneously co-circulating four Wa and two DS-1 genogroup rotaviruses were determined and analyzed together with G8P[8] rotavirus. Whole genomic phylogenetic analysis showed that these G1/G9-P[8] RVAs in this study were assigned to Wa genogroup, while those G2P[4] and G8P[8] RVAs were assigned to DS-1 genogroup. The genome constellation of six G8P[8] strains were assigned to G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2.)

L261-263, compared with previous ones conducted in China, the improvement in present study is the whole genome of simultaneously co-circulating four Wa-like G1P[8]/G9P[8] and two DS-1-like G2P[4] rotaviruses were determined and analyzed together with G8P[8] rotavirus. By analysis the relationship between Chinese G8P [8] and other local epidemic strains, the possible origin of G8P [8] is revealed.

L270-271, compared with previous ones conducted in China, we drew a different conclusion that G8P[8] rotavirus possessed the VP7 gene from bovine.

L229-235, the contents related to rotavirus vaccine were added. LLR is a G10P[15]  lamb rotavirus. It is different from the endemic human rotavirus in Wuhan. If LLR were included in the phylogenetic trees, it would become a root.

L274-276, more contents related to the significance of this study were added.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Rotavirus infections are a hot topic in clinical virology and pediatric infectious diseases. Rotaviruses remain a leading causative agent of severe dehydrating diarrhea among infants and young children. Rotavirus surveillance is established in many countries to monitor the trends in circulation, estimate the disease burden and for the countries where the rotavirus vaccine is introduced, to monitor the impact of vaccination on disease and epidemiology.

Here, the authors present a study exploring surveillance data gathered in Wuhan, China, in the period 2019-2022, i.e. the studied period covers about six months before the COVID-19 pandemic and two and a half years in the pandemic. Full genome analysis is carried out and the origin and molecular evolution of emerging G8 strains is discussed. The manuscript presents a comprehensive overview of the molecular epidemiology of rotaviruses in the epicenter of the COVID-19 pandemic during its first years and I consider it a very interesting research. The article is well-written and organized, making it easy to follow and understand. Overall, the article is a contribution to the literature on the topic.

Nevertheless, I would like to share my minor comments:

1. Line 30, line 322 – I suggest a better wording for the phrase “modern typical”. ‘Modern’ and ‘typical’ side by side are mutually exclusive.

2. Line 40 – I would suggest the omission of ‘young animals’ since the paper deals with humans only.

3. Line 41 – I would suggest a more recent reference, i.e. Du Y, Chen C, Zhang X, Yan D, Jiang D, Liu X, Yang M, Ding C, Lan L, Hecht R, Zhu C, Yang S. Global burden and trends of rotavirus infection-associated deaths from 1990 to 2019: an observational trend study. Virol J. 2022 Oct 20;19(1):166. doi: 10.1186/s12985-022-01898-9.

 

4. Line 43 – disease should be in singular.

 

5. Line 47 – please refer to the latest release of the ICTV. According to it, rotaviruses are one of the genera in Sedoreoviridae, order Reovirales. I would also suggest mentioning the 9 species (A-J) in the Rotavirus genus.

 

6. Line 112 – I suppose that ‘including’ should be ‘included’.

 

7. Line 183 – I suppose that there is a typo and instead of M, there should be R.  According to the 11-gene typing system for rotaviruses VP1 type is presented as R_x, not M_x. M stands for VP3.

 

8. Line 279-280 – it would be interesting to compare the number of samples usually gathered for the same time period in pre-pandemic times.

 

9. Line 312, line 313, line 315, line 316 – I suggest adding ‘-like’ after Wa and DS-1.

 

10. Lines 382-384 – a more detailed presentation of the procedure (or the respective reference) should be provided.

Author Response

Point-by-point response to Reviewer #2

Reviewer #2

Q1:Line 30, line 322 – I suggest a better wording for the phrase “modern typical”. ‘Modern’ and ‘typical’ side by side are mutually exclusive.

A1:Line 24, line 251: according to the suggestion, the word “modern” was deleted.

 

Q2:Line 40 – I would suggest the omission of ‘young animals’ since the paper deals with humans only.

A2: Line 32, the words “and young animals” was deleted.

 

Q3:Line 41 – I would suggest a more recent reference, i.e. Du Y, Chen C, Zhang X, Yan D, Jiang D, Liu X, Yang M, Ding C, Lan L, Hecht R, Zhu C, Yang S. Global burden and trends of rotavirus infection-associated deaths from 1990 to 2019: an observational trend study. Virol J. 2022 Oct 20;19(1):166. doi: 10.1186/s12985-022-01898-9.

A3: Line 32, according to the suggestion, the literature was cited.

 

Q4:Line 43 – disease should be in singular.

A4:Line 34, the word was corrected to ‘disease’.

 

Q5: Line 47 – please refer to the latest release of the ICTV. According to it, rotaviruses are one of the genera in Sedoreoviridae, order Reovirales. I would also suggest mentioning the 9 species (A-J) in the Rotavirus genus.

A5:Line 37-39, according to the suggestion, the presentation of taxonomy of rotavirus and the nine species of it was corrected and mentioned respectively.

 

Q6:Line 112 – I suppose that ‘including’ should be ‘included’.

A6:L108, ‘including’ was corrected to ‘included’.

 

Q7:Line 183 – I suppose that there is a typo and instead of M, there should be R.  According to the 11-gene typing system for rotaviruses VP1 type is presented as Rx, not Mx. M stands for VP3.

A7:L153, the mistake was corrected to “R2”. Similar mistakes were corrected in line 161.

 

Q8:Line 279-280 – it would be interesting to compare the number of samples usually gathered for the same time period in pre-pandemic times.

A8:It was compared and described in L79-85 and L213-221.

 

Q9:Line 312, line 313, line 315, line 316 – I suggest adding ‘-like’ after Wa and DS-1.

A9: L113, L253 and L271 ‘like’ was added after Wa and DS-1.

 

Q10:Lines 382-384 – a more detailed presentation of the procedure (or the respective reference) should be provided.

A10: L294-296 and 301-303, provided the name of kits for RT-PCR, the primer sequence used for RT-PCR, and sequencing and references as well.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

In this manuscript, Zhou et al reported human rotavirus A prevalence during the period from 2019 to 2022 in Wuhan, China. As expected, the detection rate of RVA in children is higher than in adults. Vp4 and VP7 genotyping and further phylogenetic analysis suggested that since 2021, the G8P8 genotype became the dominant genotype. Near-full genome sequencing was also performed for several RVA strains, and the results implied possible reassortments of the G8P8 strains. Overall, this report provided some useful information for RVA infection monitoring and future studies on RVA genetic diversity.

Specific comments are:

1.       Line 65-75: This description of the RVA nomenclature and classification should be moved further up, maybe before the paragraph that started at line 58. Please revise accordingly.

2.       All figures: Because of the high nucleotide sequence identities, the lines in the phylogenetic tree are largely overlapping. Please consider re-scaling the figures so that they are less crowded. 

3.       Please provide more details in the method sections 4.3 and 4.4, such as the primers used for RT and PCR amplification, and the RT-PCR kit brand.  

4.       Some typos: In line 183, “M1” should be “R2”; In line 197, “C1 and M1 genotypes” should be “C2 and M2 genotypes”.

5.       Table 1, total, adult, G9P[8]: the number “0” should be “8”.

Author Response

Point-by-point response to Reviewer #3

Q1:Line 65-75: This description of the RVA nomenclature and classification should be moved further up, maybe before the paragraph that started at line 58. Please revise accordingly.

A1: L50-58, The description of the RVA nomenclature and classification was moved before the paragraph that started as “some rotavirus strains seem to be well …”.

 

Q2:All figures: Because of the high nucleotide sequence identities, the lines in the phylogenetic tree are largely overlapping. Please consider re-scaling the figures so that they are less crowded.

A2:According to the reviewer’s advice, we reduced the number of sequences in tables and phylogenetic trees. All the phylogenetic trees were rebuilded and rescaled to make them less crowded.

 

Q3:Please provide more details in the method sections 4.3 and 4.4, such as the primers used for RT and PCR amplification, and the RT-PCR kit brand.

A3: L294-296 and 301-303, provided the name of kits for RT-PCR, the primer sequence used for RT-PCR, and sequencing and references as well.

 

Q4:Some typos: In line 183, “M1” should be “R2”; In line 197, “C1 and M1 genotypes” should be “C2 and M2 genotypes”.

A4: L153, the mistake was corrected to “R2”. Similar mistakes were corrected in line 161.

 

Q5:Table 1, total, adult, G9P[8]: the number “0” should be “8”.

A5: L100, Table 1, G9P[8] adult: the number “0” was corrected to “8”.

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I like to thank authors for their answers to my questions. At this point, I don't have more concerns about this study.