Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of this manuscript analyzed 384 patient samples and characterized 220 norovirus strains in the Sverdlovsk region in 2024. The most prevalent genotypes were GII.4 (40%), GII.7 (39%), and GII.17 (6%). Using NGS, researchers assembled eight complete genomes of the dominant GII.4[P16] and GII.7[P7] strains, discovering non-synonymous substitutions that suggest these viruses are evolving to potentially evade immune responses. This research provides crucial insights into the current genotypic composition and evolutionary dynamics of circulating noroviruses. underscoring the importance of continued genomic surveillance to predict future outbreaks. The manuscript is well written. The manuscript is well-written. However, the information in Figure 4 is also represented in Figure 5; therefore, please remove Figure 4.

 

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions.

Concerning our reply to Reviewer 1 comments, we acknowledge that figures 4 and 5 may look similar, but the significance of each tree is different. Both phylogenetic trees are complementary and underscore the phyletic relationships and the evolutionary distance of GII.4 strains.

Comments from the Editors and Reviewers:

Reviewer 1: The authors of this manuscript analyzed 384 patient samples and characterized 220 norovirus strains in the Sverdlovsk region in 2024. The most prevalent genotypes were GII.4 (40%), GII.7 (39%), and GII.17 (6%). Using NGS, researchers assembled eight complete genomes of the dominant GII.4[P16] and GII.7[P7] strains, discovering non-synonymous substitutions that suggest these viruses are evolving to potentially evade immune responses. This research provides crucial insights into the current genotypic composition and evolutionary dynamics of circulating noroviruses. underscoring the importance of continued genomic surveillance to predict future outbreaks. The manuscript is well written.

Comments 1: However, the information in Figure 4 is also represented in Figure 5; therefore, please remove Figure 4.

Response:

We thank the reviewer for this point. Two figures are presented in our paper and we prefer to retain them both. Figure 4 shows a cladogram depicting the phyletic relationships among the GII.4 taxa; this figure is necessary to represent the overall structure of the phylogram topology. However, Figure 5 shows an additive phylogram in which the length of each terminal branch represents the evolutionary distance between taxa. To preserve the overall structure and to keep the results of our publication informative, it is important to keep both figures in the manuscript. Figures 4 and 5 confirm what is written in the manuscript about the phyletic relationships and the evolutionary distance of GII.4 strains (lines 263-265; 270-276).

 

On behalf of all the authors, thank you for your kind consideration!

 

Roman O. Bykov, Postgraduate student of the Federal Budgetary Institution of Science State Scientific Center of Virology and Biotechnology "Vector" Junior Research Assistant in the Laboratory of Enteric Viral Infections. Ekaterinburg Research Institute of Viral Infections, Federal Budgetary Institution of Science «Federal Scientific Research Institute of Viral Infections «Virome» Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing Rospotrebnadzor, 620030, Ekaterinburg, Russian Federation +7(982) 690-69-64

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General overview:

The study conducted by the authors addresses an important public health topic, as human noroviruses (HuNoVs) are recognized as a leading cause of acute gastroenteritis worldwide. The work focuses on the genotypic composition of HuNoVs circulating in China during 2024, employing both Sanger sequencing and next-generation sequencing. This study makes contributions into the molecular epidemiology of HuNoVs and supports a better understanding of their role in acute diarrheal disease.

The manuscript is concise and generally well-written, although some modifications could be made to enhance its quality and comprehensibility

• Introduction:

Page 3, Lines 81–83: Figure 1 seems unnecessary, as the information is already clear in the text; if removed, please renumber the remaining figures accordingly.

• Material and Methods

Page 3, Lines 103-110: The sampling description requires further clarification. Please specify: 

• the number of municipalities in Sverdlovsk region from which patients were recruited;
• the total number of fecal samples collected and analyzed;
• whether the initial laboratory diagnosis was performed locally, and only the positive samples for NoV (n=384) forwarded to Federal Scientific Research Institute of Vital Infections to molecular characterization;
• or whether all samples were analyzed directly at Institute Federal Scientific Research Institute of Vital Infections.

These clarifications are important to better understand the representativeness of the sampling and the diagnostic workflow.

• Results

Please consider adding a table summarizing the epidemiological data of the sequenced norovirus cases (age, sex, severity of clinical manifestations when available, and genotype) to better illustrate the clinical–epidemiological context of your phylogenetic findings.

Page 8, Figure 2

In the figure, the bar for genotype GII.7\[P7] should indicate 86 samples, not 82. Please, correct this discrepancy.

Page 8, Lines 234-240     

The authors describe highly heterogeneous genetic divergence among municipalities, but the specific municipalities are not identified. It would improve clarity if the authors specify which municipalities were included in this analysis and, if considered appropriate; add a map of the region highlighting these municipalities.

Page 9, Figure 3

Please revise the legend of Figure 3 for clarity. A suggested wording is: “Reconstruction of phylogenetic events for nucleotide sequences from the VP1/RdRp fragment of genovariants GII.4\[P16] and GII.7\[P7] in 2024, using the Kimura 2-parameter model. Black squares represent genovariants GII.4\[P16], and black triangles represent genovariants GII.7\[P7] identified in the  Sverdlovsk region in 2024”.

 

• Discussion

Page 14, Lines 402-407

Consider adding a brief note on the patients’ clinical course and exploring whether the observed amino acid substitutions (e.g., in GII.4[P16]) could be related to clinical manifestations. If data on disease severity are available, incorporating this information could further highlight the potential clinical relevance of the genetic findings.

The manuscript would be strengthened by the inclusion of a brief discussion of the study’s limitations.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions.

Concerning the comments from Reviewer 2, we have addressed each separately. Please note that the table summarizing the epidemiological data proposed by reviewer 2 in point 3, has been added as a supplementary file. The table is very large to be published in the main manuscript. A new figure was added (figure 2) showcasing all the included municipalities in this study.

Comments from the Editors and Reviewers:

Reviewer 2: The study conducted by the authors addresses an important public health topic, as human noroviruses (HuNoVs) are recognized as a leading cause of acute gastroenteritis worldwide. The work focuses on the genotypic composition of HuNoVs circulating in China during 2024, employing both Sanger sequencing and next-generation sequencing. This study makes contributions into the molecular epidemiology of HuNoVs and supports a better understanding of their role in acute diarrheal disease.

The manuscript is concise and generally well-written, although some modifications could be made to enhance its quality and comprehensibility.

Comments 1: Page 3, Lines 81–83: Figure 1 seems unnecessary, as the information is already clear in the text; if removed, please renumber the remaining figures accordingly.

Response:

We thank the reviewer for this point. Figure 1 depicts a simplified representation of the structure of the major capsid protein VP1 in noroviruses, including the main antigenic determinants responsible for mediating norovirus penetration into the target cell and eliciting specific immune responses in humans. We consider it reasonable to retain Figure 1 in the manuscript to visually demonstrate the role of norovirus structural proteins in the infection process. The structural proteins and their components are cited in the introduction and the discussion. Therefore, this would allow readers that are specialists in norovirus research or scientists encountering this topic for the first time to better understand this norovirus topic.

Comments 2: Material and Methods. Page 3, Lines 103-110: The sampling description requires further clarification. Please specify:  the number of municipalities in Sverdlovsk region from which patients were recruited; the total number of fecal samples collected and analyzed; whether the initial laboratory diagnosis was performed locally, and only the positive samples for NoV (n=384) forwarded to Federal Scientific Research Institute of Vital Infections to molecular characterization; or whether all samples were analyzed directly at Institute Federal Scientific Research Institute of Vital Infections. These clarifications are important to better understand the representativeness of the sampling and the diagnostic workflow.

Response:

We thank the reviewer for this important comment. In 2024, the collection of stool samples from patients with a confirmed diagnosis of HuNoVs was organized in four municipalities: Ekaterinburg, Kamensk-Uralsky, Nizhny Tagil, and Sukhoy Log. The total number of “positive” biological samples collected during the 2024 analytical period was n=384. Samples submitted to our institute were screened by either an enzyme immunoassay or qPCR at two medical facilities: The Ekaterinburg Consultative Diagnostic Center and the State Autonomous Healthcare Institution the Sverdlovsk Region Children’s City Hospital (Kamensk-Uralsky). Figure 2 was added as proposed in comment 5.

This was added to the manuscript (lines 106-116), and the text now read:

«The collection of stool samples from patients with a confirmed diagnosis of HuNoVs was organized in four municipalities during 2024: Ekaterinburg, Kamensk-Uralsky, Nizhny Tagil, and Sukhoy Log. Samples from patients were screened for HuNoVs by either an enzyme immunoassay (Norovirus-antigen-enzyme immunoassays-Best, Vector-Best, Novosibirsk, Russian Federation) or qPCR (AmpliSens® Norovirus GI-GII-FL, Central Scientific Research Institute of Epidemiology, Moscow, Russian Federation) at the following facilities: The Ekaterinburg Consultative Diagnostic Center and the State Autonomous Healthcare Institution the Sverdlovsk Region Children’s City Hospital (Kamensk-Uralsky). Only positive samples were submitted to the Federal Scientific Research Institute of Viral Infections «Virome» (n=384) for further molecular analysis»

Comments 3: Please consider adding a table summarizing the epidemiological data of the sequenced norovirus cases (age, sex, severity of clinical manifestations when available, and genotype) to better illustrate the clinical–epidemiological context of your phylogenetic findings.

Response: We have revised the table in accordance with the comments provided. Due to the large volume of data, it was decided to add the table to the supplementary materials section.

Table in the supplementary materials section. Data on identified cases of norovirus infection. (sample number, sex, age and genotype)

Number	Sex	Age	Genotype
2	-	2	GII.4[P16]
4	-	5	GII.4[P16]
9	-	5	GII.4[P16]
10	-	3	GII.4[P16]
11	-	5	GII.4[P16]
13	-	4	GII.4[P16]
14	-	3	GII.4[P16]
15	-	3	GII.4[P16]
16	-	2	GII.4[P16]
27	-	3	GII.4[P16]
61	-	5	GII.4[P16]
63	male	13	GII.4[P16]
79	-	3	GII.4[P16]
83	-	4	GII.4[P16]
84	-	9	GII.4[P16]
92	-	3	GII.4[P16]
93	-	3	GII.4[P16]
102	male	3	GII.4[P16]
105	female	5	GII.4[P16]
107	female	12	GII.4[P16]
108	female	13	GII.4[P16]
110	female	5	GII.4[P16]
111	male	4	GII.4[P16]
154	female	-	GII.4[P16]
155	female	-	GII.4[P16]
159	female	8	GII.4[P16]
163	male	3	GII.4[P16]
190	male	-	GII.4[P16]
192	male	7	GII.4[P16]
201	female	3	GII.4[P16]
208	female	12	GII.4[P16]
210	female	-	GII.4[P16]
231	male	6	GII.4[P16]
251	male	6	GII.4[P16]
253	male	3	GII.4[P16]
254	female	-	GII.4[P16]
259	female	15	GII.4[P16]
383	-	-	GII.4[P16]
384	-	-	GII.4[P16]
390	-	-	GII.4[P16]
400	female	23	GII.4[P16]
401	female	8	GII.4[P16]
402	female	3	GII.4[P16]
403	male	8	GII.4[P16]
404	female	-	GII.4[P16]
405	male	13	GII.4[P16]
456	male	4	GII.4[P16]
457	male	3	GII.4[P16]
459	-	11	GII.4[P16]
500	-	3	GII.4[P16]
501	-	4	GII.4[P16]
508	-	5	GII.4[P16]
511	-	16	GII.4[P16]
513	-	2	GII.4[P16]
514	-	2	GII.4[P16]
602	-	2	GII.4[P16]
604	-	5	GII.4[P16]
606	-	3	GII.4[P16]
608	-	2	GII.4[P16]
609	-	3	GII.4[P16]
612	-	12	GII.4[P16]
614	-	1	GII.4[P16]
619	-	2	GII.4[P16]
680	female	4	GII.4[P16]
687	male	6	GII.4[P16]
690	male	4	GII.4[P16]
698	male	3	GII.4[P16]
707	male	15	GII.4[P16]
708	female	2	GII.4[P16]
819	-	6	GII.4[P16]
1001	-	5	GII.4[P16]
1107	male	6	GII.4[P16]
1213	female	6	GII.4[P16]
1264	-	3	GII.4[P16]
1266	-	3	GII.4[P16]
1272	-	3	GII.4[P16]
1273	-	4	GII.4[P16]
1275	-	2	GII.4[P16]
1276	-	2	GII.4[P16]
1629	female	3	GII.4[P16]
1630	female	3	GII.4[P16]
1633	female	3	GII.4[P16]
1638	male	4	GII.4[P16]
1640	female	4	GII.4[P16]
1679	-	4	GII.4[P16]
1680	-	2	GII.4[P16]
1850	-	4	GII.4[P16]
1987	-	6	GII.4[P16]
1	-	3	GII.7[P7]
8	-	2	GII.7[P7]
26	-	3	GII.7[P7]
28	male	7	GII.7[P7]
29	male	3	GII.7[P7]
30	female	9	GII.7[P7]
31	female	11	GII.7[P7]
34	female	11	GII.7[P7]
36	female	9	GII.7[P7]
37	male	9	GII.7[P7]
38	female	-	GII.7[P7]
39	female	-	GII.7[P7]
40	female	-	GII.7[P7]
41	female	-	GII.7[P7]
42	male	21	GII.7[P7]
43	male	8	GII.7[P7]
44	female	10	GII.7[P7]
45	female	4	GII.7[P7]
46	female	5	GII.7[P7]
64	-	16	GII.7[P7]
65	-	19	GII.7[P7]
66	-	6	GII.7[P7]
67	-	12	GII.7[P7]
68	male	5	GII.7[P7]
78	-	4	GII.7[P7]
85	-	9	GII.7[P7]
87	-	15	GII.7[P7]
88	-	12	GII.7[P7]
90	-	12	GII.7[P7]
106	female	3	GII.7[P7]
156	female	4	GII.7[P7]
157	female	3	GII.7[P7]
158	male	10	GII.7[P7]
161	female	11	GII.7[P7]
162	-	3	GII.7[P7]
164	female	10	GII.7[P7]
165	male	17	GII.7[P7]
166	male	4	GII.7[P7]
168	female	3	GII.7[P7]
171	female	14	GII.7[P7]
175	female	8	GII.7[P7]
177	male	2	GII.7[P7]
178	female	9	GII.7[P7]
179	female	3	GII.7[P7]
180	male	7	GII.7[P7]
181	female	4	GII.7[P7]
186	male	3	GII.7[P7]
191	male	4	GII.7[P7]
200	-	2	GII.7[P7]
202	female	11	GII.7[P7]
211	male	3	GII.7[P7]
215	female	5	GII.7[P7]
218	female	6	GII.7[P7]
219	female	6	GII.7[P7]
220	female	2	GII.7[P7]
222	female	10	GII.7[P7]
223	-	4	GII.7[P7]
224	male	7	GII.7[P7]
228	female	8	GII.7[P7]
233	-	3	GII.7[P7]
252	male.	3	GII.7[P7]
256	male	18	GII.7[P7]
258	male	9	GII.7[P7]
261	-	-	GII.7[P7]
263	-	6	GII.7[P7]
267	-	21	GII.7[P7]
268	male	9	GII.7[P7]
269	male	13	GII.7[P7]
271	female	8	GII.7[P7]
274	female	21	GII.7[P7]
276	male	5	GII.7[P7]
277	female	5	GII.7[P7]
278	male	4	GII.7[P7]
279	female	3	GII.7[P7]
283	male	4	GII.7[P7]
284	-	4	GII.7[P7]
453	male	17	GII.7[P7]
454	male	7	GII.7[P7]
460	male	12	GII.7[P7]
461	male	16	GII.7[P7]
462	female	10	GII.7[P7]
464	male	10	GII.7[P7]
610	-	3	GII.7[P7]
620	female	14	GII.7[P7]
622	female	1	GII.7[P7]
627	male	7	GII.7[P7]
628	-	3	GII.7[P7]
629	-	2	GII.7[P7]
25	-	5	GII.17[P17]
387	-	-	GII.17[P17]
388	male	-	GII.17[P17]
389	-	-	GII.17[P17]
475	-	-	GII.17[P17]
611	-	12	GII.17[P17]
659	-	1	GII.17[P17]
823	female	12	GII.17[P17]
824	female	9	GII.17[P17]
825	-	15	GII.17[P17]
827	-	14	GII.17[P17]
1220	female	1	GII.17[P17]
1223	-	13	GII.17[P17]
1692	-	2	GII.17[P17]
59	female	2	GIV.1
234	-	17	GI.2[P2]
241	-	12	GI.2[P2]
250	female	10	GI.2[P2]
1860	-	3	GI.2[P2]
1873	female	2	GI.2[P2]
1877	male	2	GI.2[P2]
1881	female	5	GI.2[P2]
1761	female	3	GI.2[P2]
1762	-	9	GI.7[P7]
1763	male	10	GI.7[P7]
1766	-	11	GI.7[P7]
1768	female	8	GI.7[P7]
1769	female	13	GI.7[P7]
70	-	2	GII.6[P7]
71	male	1	GII.6[P7]
184	female	6	GII.6[P7]
199	-	3	GII.6[P7]
72	-	-	GII.2[Px]
75	-	2	GII.3[P12]
80	-	5	GII.3[P12]
422	-	19	GII.3[P12]
423	female	19	GII.3[P12]
477	-	-	GII.3[P12]
479	-	-	GII.3[P12]
820	-	18	GII.3[P12]
73	-	2	GII.3[P12]
172	male	-	GI.6[P7]
174	-	-	GI.6[P7]
176	-	-	GI.6[P7]

 

 

Comments 4: Page 8, Figure 2. In the figure, the bar for genotype GII.7\[P7] should indicate 86 samples, not 82. Please, correct this discrepancy

 

Response:

We thank the reviewer for his careful reading of our manuscript. We made changes to Figure 8.

 

Comments 5: Page 8, Lines 234-240. The authors describe highly heterogeneous genetic divergence among municipalities, but the specific municipalities are not identified. It would improve clarity if the authors specify which municipalities were included in this analysis and, if considered appropriate; add a map of the region highlighting these municipalities.

Response:

Thank you for the suggestion. A figure (Figure 2) showing the main municipalities that participated in the study, has been added to the main text of the manuscript.

 

Figure 2. Municipalities of the Sverdlovsk region participating in this study (1—Ekaterinburg, 2— Kamensk-Uralsky, 3— Nizhny Tagil, 4—Sukhoy Log). 

Comments 6: Page 9, Figure 3. Please revise the legend of Figure 3 for clarity. A suggested wording is: “Reconstruction of phylogenetic events for nucleotide sequences from the VP1/RdRp fragment of genovariants GII.4\[P16] and GII.7\[P7] in 2024, using the Kimura 2-parameter model. Black squares represent genovariants GII.4\[P16], and black triangles represent genovariants GII.7\[P7] identified in the Sverdlovsk region in 2024”.

Response:

Thank you for this valuable comment. The caption for Figure 3 was revised and now reads:

«Reconstruction of phylogenetic events for nucleotide sequences from the VP1/RdRp fragment of genovariants GII.4[P16] and GII.7[P7] in 2024, using the Kimura 2-parameter model. Black squares represent genovariants GII.4[P16], and black triangles represent genovariants GII.7[P7] identified in the  Sverdlovsk region in 2024»

Comments 7: Discussion

Page 14, Lines 402-407. Consider adding a brief note on the patients’ clinical course and exploring whether the observed amino acid substitutions (e.g., in GII.4[P16]) could be related to clinical manifestations. If data on disease severity are available, incorporating this information could further highlight the potential clinical relevance of the genetic findings.

Response:

The study sample lacks information on the severity of the clinical course. Unfortunately, we cannot interpret the impact of mutations in VP1 antigenic determinants on disease severity.

Comments 8: The manuscript would be strengthened by the inclusion of a brief discussion of the study’s limitations.

Response:

One of this study’s limitation is the choice of method for genotyping. Different laboratories may use varying methods and protocols for norovirus genotyping, leading to significant differences in results and reducing the overall reliability and comparability of genotyping data. Another limitation in our study is the number norovirus strains that were included in the NGS analysis. We choose five GII.4 strains and five GII.7 strains as the most prevalent HuNoV genotype in our region. We could not include more HuNoV strains due to financial limitations and the need to achieve a better genome coverage by NGS. Due to same reasons, we did not include in the full-genome analysis the less prevalent circulating genotypes. 

The brief discussion of the study’s limitations has been added (lines 429-437):

«One of the main limitation in HuNoV genotyping is the choice of method. Different laboratories may use varying methods and protocols for, leading to significant differences in results and reducing the overall reliability and comparability of genotyping data. The main limitation in our study is the number of included norovirus strains in the NGS analysis. We choose five GII.4 strains and five GII.7 strains, as the most prevalent and representative HuNoV genotypes in our region. We could not include more HuNoV strains due to financial limitations and the need to achieve a better genome coverage by NGS. Due to same reasons, we did not include the less prevalent circulating genotypes despite the vast diversity of HuNoV genotypes in our results.»

On behalf of all the authors, thank you for your kind consideration!

Roman O. Bykov, Postgraduate student of the Federal Budgetary Institution of Science State Scientific Center of Virology and Biotechnology "Vector" Junior Research Assistant in the Laboratory of Enteric Viral Infections. Ekaterinburg Research Institute of Viral Infections, Federal Budgetary Institution of Science «Federal Scientific Research Institute of Viral Infections «Virome» Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing Rospotrebnadzor, 620030, Ekaterinburg, Russian Federation +7(982) 690-69-64

 

 

 

 

 

 

 

Author Response File: Author Response.pdf