Is ZIKA Virus re-emerging as a distinct genetic lineage in India?
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An outbreak of Zika fever occurred in Thiruvananthapuram City, Kerala, India during 2021. On the request of Kerala state health administration, we investigated the same, towards proposing requisite containment strategies of the disease outbreak. Epidemiological investigations indicated a clustering pattern of Zika cases with the presumed index case from a multi-speciality hospital in the city. Preliminary reports on the same had been already reported elsewhere during 2021. Further, entomological surveys carried out evinced the predominant mosquito species in the City viz., Aedes albopictus (65.55%), Aedes aegypti (22.0%) and Aedes vittatus (12.0%) were naturally infected with Zika Virus (ZIKV), the Minimum Infection Rates (MIR) being 17.9, 7.8 and 3.6 respectively. Also, the trans-ovarian transmission was recorded in Ae. albopictus. Analysis of phylogenetically informative genes of ZIKV genome indicated the emergence of a distinct lineage of the Asian strain of the virus with four unique non-synonymous mutations viz., “I22T” & “I160M” (pre-Membrane) and “D348N” & “V473L” (Envelope) was involved in the outbreak. The altered gene expression pattern and evolutionary implications of these unique mutations remain to be investigated. Genetic analysis of the virus isolates from this and other investigations carried out on sporadic outbreaks of ZIKV in the country subsequently, indicated ZIKV is reemerging as a distinct genetic lineage in India. These findings warrant the urgent need for a systematic country-wide surveillance strategy, towards the prevention/ preparedness/ containment of a massive outbreak of this emerging neurovirulent arboviral disease.
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Dear AISWARYA R S Thank you for your submission, following reviews I have seldcted Major revisions for the manuscript. Authors raised concerns around inclusion of methodologically relevent information and general concerns around formatting, grammer and inclusion of information in specific sections. Please address the reviewers concerns and any questions please dont hesitate to reach out. Best wishes, John.
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Comments to Author
The manuscript described recent findings of a distinct ZIKV lineage emergence in India based on analysis capsid-pre-membrane, envelope and non-structural protein 5 amino acid sequences. The manuscript need some improvements to give more evidence and clear description to the audience. General: 1. Please be consistent on writing Zika virus. When the topic describing Zika virus infection, please write it clearly. For example line 207-208 can be written as 'ZIKV-infected patient' instead of 'Zika-infected patient'. Also, line 213-217 there are three different writing styles: Zika, ZIKV and ZIKA. 2. Please follow the taxonomy guideline to write animal species. - Introduction Line 90: The virus nomenclature has changed, please use the new nomenclature e.g. genus Flavivirus has changed to Orthoflavivirus. …
Comments to Author
The manuscript described recent findings of a distinct ZIKV lineage emergence in India based on analysis capsid-pre-membrane, envelope and non-structural protein 5 amino acid sequences. The manuscript need some improvements to give more evidence and clear description to the audience. General: 1. Please be consistent on writing Zika virus. When the topic describing Zika virus infection, please write it clearly. For example line 207-208 can be written as 'ZIKV-infected patient' instead of 'Zika-infected patient'. Also, line 213-217 there are three different writing styles: Zika, ZIKV and ZIKA. 2. Please follow the taxonomy guideline to write animal species. - Introduction Line 90: The virus nomenclature has changed, please use the new nomenclature e.g. genus Flavivirus has changed to Orthoflavivirus. The complete new nomenclature can be found at ictv.global Line 102: Pandemic is used to described a sudden increase of cases spreading in several countries, continents or whole world while epidemic is a sudden increase of cases in a large population and outbreak is similar to epidemic but in a smaller population. In this case, might be better to describe the cases as outbreaks. Line 119: Indonesia and Malaysia are two different countries in Southeast Asia. The 1966 ZIKV was isolated in Malaysia, not Indonesia. Indonesia reported the first human case in Central Java in 1977. -Material and methods: General: Please separate into several subheadings, e.g. (1) Study area; (2) Clinical case; (3) Entomological survey; (4) ZIKV molecular detection; (5) Data analysis. Line 170: What is 'immature mosquito'? Is it pupae or eggs? Line 173-174: Please describe the methodology for mosquito identification. What is the guideline do you use the distinguish between mosquito species? Line 174-175: Please decribe in detail the methodology for the ZIKV detection in mosquito samples by real-time RT-PCR. Line 176-177: Please describe in detail about human samples and the methodology to screen ZIKV from those samples. Line 191: What is the specificity and sensitivity of the primer sets? Do you used the sampe primer sets to screen ZIKV in mosquito and human samples? - Results Epidemiological investigations Line 211: What is ZIKA symptoms in this context? Please explain in details. Entomological studies General: What is the total number of collected mosquitoes? Line 222: What are the immature specimens? Line 227: a) The total number described in line 221 is 2216 with 1643 immatures and 573 adult specimens. What is 1466 specimens mentioned in this line refer to? b) Please described the 261 pools (species-wise) clearly (How many pools do you have per species?). Line 228-229: Please describe the conventional diagnostic PCR and real-time PCR protocol in materials and methods section. Line 223-234: Please describe the methodology to calculate MIR in materials and methods section. Line 237-238: Please described the results of ZIKV screening in human samples separately. Genetic Characterization of the virus General: a) What is the differences of Diagnostic marker sequences and Non-structural Protein 5 gene? Both sections describing the mutation in the ZIKV NS5 region, why do these two sections need to be separated? b) For each genes, please describe the phylogenetic tree first then explain the detail mutations. c) For the ZIKV genome analysis, please include ZIKV sequences from India and other countries in South Asia before 2018. d) Focus to explain the findings or results first before make a relation to the references or published data. Diagnostic marker sequences: Line 243: a) Please describe the methodology for mosquito rearing in the Materials and Methods section. b) How do you rule out the possiblity of cross-contamination in male mosquito samples? Line 246: Which nucleotide is changed? If it is adenine to guanine? If so, it can described as "...synonymous mutation from adenine to guanine at position 1512 (A1512G)". Line 247: a) Please describe the total number of unfed, semi-gravid and gravid mosquitoes collected in this study. b) What is 'unfed' in this context? Is it not-blood fed? If so, please mention the total number (and pools) of blood-fed and not-blood-fed mosquitoes. Capsid - pre-Membrane Gene: Line 258-260: Please include the gene reference number for the reference genome used for comparison. Envelope gene: Line 277-279: Please rephrase the sentence to make it more clear and straight forward to the audience. Non-structural Protein 5 gene: Line 286-288: Why M114V mutation is important in American lineage? Please explain it clearly. -Discussion: Are there any surveillance data that showed that the ZIKV is re-emerging in India recently? - Figures Figure 2: Please provide a legend to describe the color code that used in the Venn diagram. Figure 4: a) ZIKV American and Indian lineages are part of Asian lineage, please provide the accession number of ZIKV sequence that used for the comparison. b) The mutations in the C-prM, E and NS5 proteins will be much clearer when you show the amino acid sequence allignments and highlight the point mutations.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
No: They have got the ethical approval to use human samples, but they did not explain clearly in the materials and metholody section on what and how they collected the samples, storage, processing and disposal. Due to the detail information is not available, I do not know how they handle and human samples and either their handling is approproate or not.
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Comments to Author
The authors of this manuscript set out to describe their epidemiological and entomological survey of a recent ZIKV outbreak in an Indian City. They provide an oral description of how they retrospectively assed human sera for ZIKV after an initial positive in 2021. They then outline the entomological surveys that took place to identify Aedes albopictus as a dominant vector. Through sequencing of 3 genes they suggest a new ZIKV lineage arose in India that differs from the African and Asian lineages commonly discussed in literature. The authors show clear data demonstrating a recent ZIKV outbreak in this city, which is important as we continue to see vector-borne diseases emerging in new areas. Although this paper shows important work in the field of vector-biology, it lacks whole genome sequencing and …
Comments to Author
The authors of this manuscript set out to describe their epidemiological and entomological survey of a recent ZIKV outbreak in an Indian City. They provide an oral description of how they retrospectively assed human sera for ZIKV after an initial positive in 2021. They then outline the entomological surveys that took place to identify Aedes albopictus as a dominant vector. Through sequencing of 3 genes they suggest a new ZIKV lineage arose in India that differs from the African and Asian lineages commonly discussed in literature. The authors show clear data demonstrating a recent ZIKV outbreak in this city, which is important as we continue to see vector-borne diseases emerging in new areas. Although this paper shows important work in the field of vector-biology, it lacks whole genome sequencing and requires editing before it can be published in a scientific journal. Question - I understand that only 3 genes were amplified and sequenced throughout the entire genome. This seems like oversight by the authors given that previous research has shown mutations in the nonstructural protein besides NS5 to play a large role in viral evolution. Have the authors gone back and sequenced the rest of the genome? It seems less convincing that you discovered a new ZIKV lineage when you only sequenced 3 genes out of the whole genome. - Have the authors considered other factors that might drive the outbreak besides the specific mutations, climatic or socioeconomic factors? - In figure 1 the authors identify locations with ZIKV cases, is this positive human sera, so where the positive patient lived? The authors could be more descriptive in their figure legends - How did the work of this manuscript help contain the outbreak? The authors state in the abstract that the identification of ZIKV allowed for containment of the outbreak, but never state how. - How was the MIR calculated? The authors point it out for each species found positive for ZIKV but then never discuss how they calculated it and they do not list the different MIR in Table 2. - The authors discuss significant bootstrap values in the discussion section of the manuscript but never in the results. Is it actually significant to your manuscript? if so it should be added to the results. Comments - Table 2 is mentioned earlier in the manuscript then table 1, authors should consider renumbering - The results section is filled with information that is more appropriately placed in either the methods or discussion sections(e.g. accession numbers and function of mutations). Additionally, there are numerous section of this manuscript where the information is better suited elsewhere in the paper. - I recommend the authors make a new table outlining the accession numbers for samples discussed in this manuscript - Part of the results section is spent listing all the different mutations, but there is no table outlining them, authors should add table with Amino acid changes in addition to the figure - The authors wait till the end of the paper to mention that Aedes albopictus was found as a new vector of ZIKV in India during this outbreak. The authors should put more effort into outlining previous vector surveillance results in India prior to the 2021 outbreak. If this is the first time Ae. Albopictus pools were ZIKV positive then that point should be emphasized better.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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