The evolutionary and molecular history of a chikungunya virus outbreak lineage

In 2018–2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018–2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines.


Methods
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?
Reviewer #1: The study is designed for multi-year phylogenetic analysis of CHIKV using available public data The authors sequenced 12 additional CHIKV sequence from travelers returning from Thailand.The number of samples used for the analysis is sufficient.Conclusions are very well supported by the analysis and statistical analysis employed.

Authors' response:
We thank both reviewers for the time taken to review and provide constructive feedback to our manuscript.We have tried our best to address all concerns and comments raised.Please see below a response to all comments and the corresponding change in the manuscript.

Results
-Does the analysis presented match the analysis plan? -Are the results clearly and completely presented?-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #1: The analysis matches with the plan to describe the evolutionary of CHIKV outbreak.Figures and results are also not that clear (see below).

Authors' response:
Thank you for the comments.Please see specific comment below regarding the figures and changes made.

Conclusions
-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?
Reviewer #1: Conclusions seem to be supported by the data.The authors, however, did not mention any limitations of the analysis.

Authors' response:
We agree that the limitations of our study are not made clear enough.We have therefore added more text on this in the materials and methods section of the evolutionary and mutational analyses sections to highlight limitations.
Reviewer #1: There are some minor revisions to improve the readability.1. Please write in more detail how the sequences were assembled.

Authors' response:
We understand your suggestion to separate the figures into two.However, we believe that keeping the figures together better preserves the context and connection between the data.To enhance clarity and facilitate reference in the text, we have divided the figure into parts A and B. We hope this approach addresses your concerns while maintaining the integrity of the information.
3. There seems to be detachment of the map of the virus movement and its explanation in the manuscript.For example, line 212 says, "islands (Madagascar, Mauritius, Mayotte, Comoros) in the 1960s and to India in the 1980s (blue)", however the map shows that the year of the spread to India is 2003.Also, line 210 says, "Central Africa to the Americas (red) in the late 1980s and early 2010s (1995 in the USA and 2014", however the map shows that the spread to the USA in 1979 and to Brazil in 2013.

Authors' response:
Thank you for pointing this out.The map here shows the correct year during which the movement occurred.The correct sentence for line 212 has been corrected to: "…islands (Madagascar, Mauritius, Mayotte, Comoros) in the 1960s and to India in the early 21 st century (blue)." We also agree that the sentence on line 210 is confusing.This stems from the fact that we could not verify that the sequence "USA|KY575574.1|1995" is a mosquito collected in the US or a human infected in the US, but rather a person infected in an African country (i.e. an imported case to the US).We have tried to clear up the description and made sure that the date in the map corresponds to what is written in the text. 1 shows red arrow pointing from Cameroon to Madagascar.Is it not blue arrow?Also, the arrow pointing the movement of ECSA2 to ECSA1 from Uganda to Senegal in 1963 does not seem to fit the phylogenetic tree.

Authors' response:
The introduction from Cameroon to Madagascar, identified as the switch from ECSA2 to IOL, precedes the first occurrence of a sequence identified as IOL.Since first IOL occurrence, outbreak, and spread was documented in the Indian ocean islands, and arrow in question shows the origins to the Cameroonian ancestor, we chose to depict this in red to highlight the ancestor origine.Since no IOL strains have been detected to circulate in Cameroon and surroundings, it is likely that the mutations dividing the ancestral strain and the new IOL strains occurred either on the way from the continent to the islands or on the islands.Hence the colour choice.The green arrow, pointing to Senegal in 1963, should originate from Tanzania.We acknowledge that it previously appeared as if the arrow originated in Uganda, and this has now been adjusted for clarity.
Reviewer #2: Abstract: 1) "…we also identified amino acid substitutions that may be associated 14 with immune evasion, increased spread, and virulence."and the next sentence "…which are highly relevant as they may 16 lead to changes in vector competence, transmission efficiency and pathogenicity of the virus" are duplicates.Rephrase, please.

Authors' response:
Thank you for pointing this out.This has now been changed.