Emergence and Spread of the SARS-CoV-2 Variant of Concern Delta across Different Brazilian Regions

ABSTRACT The SARS-CoV-2 variant of concern (VOC) Delta was first detected in India in October 2020. The first imported cases of the Delta variant in Brazil were identified in April 2021 in the southern region, followed by more cases in different regions during the following months. By early September 2021, Delta was already the dominant variant in the southeastern (87%), southern (73%), and northeastern (52%) Brazilian regions. This study aimed to understand the spatiotemporal dissemination dynamics of Delta in Brazil. To this end, we employed a combination of maximum likelihood (ML) and Bayesian methods to reconstruct the evolutionary relationship of 2,264 VOC Delta complete genomes (482 from this study) recovered across 21 of the 27 Brazilian federal units. Our phylogeographic analyses identified three major transmission clusters of Delta in Brazil. The clade BR-I (n = 1,560) arose in Rio de Janeiro in late April 2021 and was the major cluster behind the dissemination of the VOC Delta in the southeastern, northeastern, northern, and central-western regions. The AY.101 lineage (n = 207) that arose in the Paraná state in late April 2021 and aggregated the largest fraction of sampled genomes from the southern region. Lastly, the AY.46.3 lineage emerged in Brazil in the São Paulo state in early June 2021 and remained mostly restricted to this state. In the rapid turnover of viral variants characteristic of the SARS-CoV-2 pandemic, Brazilian regions seem to occupy different stages of an increasing prevalence of the VOC Delta in their epidemic profiles. This process demands continuous genomic and epidemiological surveillance toward identifying and mitigating new introductions, limiting their dissemination, and preventing the establishment of more significant outbreaks in a population already heavily affected by the COVID-19 pandemic. IMPORTANCE Amid the SARS-CoV-2 continuously changing epidemic profile, this study details the space-time dynamics of the emergence of the Delta lineage across Brazilian territories, pointing out its multiple introductions in the country and its most prevalent sublineages. Some of these sublineages have their emergence, alongside their genomic composition and geographic distribution, detailed here for the first time. A special focus is given to the emergence process of Delta outside the country’s south and southeast regions, the most populated and subjects of most published SARS-CoV-2 studies in Brazil. In summary, the study allows a better comprehension of the evolution process of a SARS-CoV-2 lineage that would be associated with a significant recrudescence of the pandemic in Brazil.

The work aims to characterize the spread and circulation of the Delta variant in Brazil. The study is comprehensive, highlights regional differences, and determines specific mutations in Brazil's lineages. There are, however, some concerns about some of the strategies utilized, particularly in the subsampling.

Major concerns:
The Delta dataset's downsizing strategy consists of automatically eliminating duplicates and keeping the sequences more similar to those from Brazil. However, there is a concern about the evenness of this subset's temporal and geographical distribution, mainly since it is used to infer the geographical origin of the main sublineages. It will be helpful to show the available Delta sequences in the database by date and region and consider enriching the representation of those states with fewer samples. Given the unevenness of the sampling, it will also be helpful to present the total number of sequences available from Brazil in this period to assess if the contribution of other circulating variants was more significant in the north of Brazil or if a lack of sampling is to blame.
Given the sampling biases, what are the effective sample sizes reported by Tracer? Are they within the accepted values?
Minor concerns: There are some issues with the language in the abstract, where the verbs used are not always exact; for instance, the use of the verb "born" to mean originated should be changed The authors explored the emergence and spread of Delta in different Brazilian regions, and identified three major clusters. Although this work is important and the methods are solid, there are a few points that should be addressed in order to make the manuscript stronger.
As the authors mentioned in the introduction, until the summer of 2021 gamma was the main variant spreading through the population in Brazil. It would be of great help for the readers of this article to see a plot of variants in Brazil overtime, and show when exactly gamma was displaced by delta. This would give an overview of the state of the population when delta came to play. Moreover, linked to gamma infections, it is conceivable that the population was infected at a high rate with gamma when delta came through. What is the seropositivity in the population at the time of delta surge? Vaccination status and number cases? This information need to be there.
It would also be very useful to add to the figures how many genomes have been sequenced from each region represented and to be specific whether there is a geographic bias in sampling and how the authors overcome that limitation.
The authors should also explore the possibility of these genomes to be linked to external introductions in Brazil, for that I would suggest the authors to blast their genomes against GISAID non-Brazil genomes and collect the ones that are the most similar before and after the collection date of each genome within a month. That way it would be possible to see how these clusters are local or due to importations. This is an important analysis that needs to be made in order to be sure that the clusters the authors found are correct.
Are the clusters specific genomic signatures indication of new delta sub-lineages?
Staff Comments:

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Reviewer comments
Reviewer #1 (Public repository details (Required)): The study includes the sequencing of viral samples. Therefore, those sequences must be deposited in a public database

All Brazilian sequences newly generated by the COVID-19 Fiocruz Genomic
Surveillance network, were submitted to the GISAID database (https://www.gisaid.org/) in advance to the original submission, and their accession codes were listed in the APPENDIX 2. The other sequences, inside and outside Brazil were also downloaded from GISAID.
Reviewer #1 (Comments for the Author): 1. The work aims to characterize the spread and circulation of the Delta variant in Brazil. The study is comprehensive, highlights regional differences, and determines specific mutations in Brazil's lineages. There are, however, some concerns about some of the strategies utilized, particularly in the subsampling.
Major concerns: 2. The Delta dataset's downsizing strategy consists of automatically eliminating duplicates and keeping the sequences more similar to those from Brazil. However, there is a concern about the evenness of this subset's temporal and geographical distribution, mainly since it is used to infer the geographical origin of the main sublineages. It will be helpful to show the available Delta sequences in the database by date and region and consider enriching the representation of those states with fewer samples. 3. Given the unevenness of the sampling, it will also be helpful to present the total number of sequences available from Brazil in this period to assess if the contribution of other circulating variants was more significant in the north of Brazil or if a lack of sampling is to blame.
In order to clarify the relative prevalence of the differently circulating variants across time, we added a new Fig 1, showing the contribution of Delta (B.1.617.2 + AY*), Gamma (P.1 + P.1*) and other circulating variants in the country's available genome sequences. In   Fig 1 A-F  To acquiesce the reviewer's concern, the methodology section was updated to clarify this aspect.
Minor concerns: 5. There are some issues with the language in the abstract, where the verbs used are not always exact; for instance, the use of the verb "born" to mean originated should be changed The abstract text was revised in compliance with the reviewer's suggestion.
6. Figure 1 legend: please correct "their dimension have the same scholar scheme".

The legend was revised in compliance with the reviewer's suggestion.
Reviewer #2 (Public repository details (Required)): 1. sars-cov-2 genomes Reviewer #2 (Comments for the Author): The authors explored the emergence and spread of Delta in different Brazilian regions, and identified three major clusters. Although this work is important and the methods are solid, there are a few points that should be addressed in order to make the manuscript stronger. 3. It would also be very useful to add to the figures how many genomes have been sequenced from each region represented and to be specific whether there is a geographic bias in sampling and how the authors overcome that limitation.  4. The authors should also explore the possibility of these genomes to be linked to external introductions in Brazil, for that I would suggest the authors to blast their genomes against GISAID non-Brazil genomes and collect the ones that are the most similar before and after the collection date of each genome within a month. That way it would be possible to see how these clusters are local or due to importations. This is an important analysis that needs to be made in order to be sure that the clusters the authors found are correct.

The sequences listed in
The way in which the dataset composition was described in the original submission was ambiguous and didn't reflect the actual process, having been updated in the newly submitted version. Therefore, it's possible the reviewer concern has already been addressed.