Data and R code used in epidemiological analyses.

Since 2014, Brazil has experienced an unprecedented epidemic caused by chikungunya virus (CHIKV), with several waves of East-Central-South-African (ECSA) lineage transmission reported across the country. In 2018, Rio de Janeiro state, the third most populous state in Brazil, reported 41% of all chikungunya cases in the country. Here we use evolutionary and epidemiological analysis to estimate the timescale of CHIKV-ECSA-American lineage and its epidemiological patterns in Rio de Janeiro. We show that the CHIKV-ECSA outbreak in Rio de Janeiro derived from two distinct clades introduced from the Northeast region in mid-2015 (clade RJ1, n = 63/67 genomes from Rio de Janeiro) and mid-2017 (clade RJ2, n = 4/67). We detected evidence for positive selection in non-structural proteins linked with viral replication in the RJ1 clade (clade-defining: nsP4-A481D) and the RJ2 clade (nsP1-D531G). Finally, we estimate the CHIKV-ECSA’s basic reproduction number (R0) to be between 1.2 to 1.6 and show that its instantaneous reproduction number (Rt) displays a strong seasonal pattern with peaks in transmission coinciding with periods of high Aedes aegypti transmission potential. Our results highlight the need for continued genomic and epidemiological surveillance of CHIKV in Brazil, particularly during periods of high ecological suitability, and show that selective pressures underline the emergence and evolution of the large urban CHIKV-ECSA outbreak in Rio de Janeiro.</div

BEAST xml files and outputs generated in this study.

BEAST xml files and outputs generated in this study.</p

Input and output files of selection analysis with Datamonkey.

Input and output files of selection analysis with Datamonkey.</p

TreeTime and HyPhy analysis.

(A) The molecular clock tree had its branches colored according to the ancestral location’s reconstructions. Tip shapes indicate sequences generated in this study. Light gray ticks along branches indicate synonymous mutations, while dark gray ticks mark non-synonymous mutations. Mutations for which evidence of positive selection was found (MEME and FEL models) are marked in red (NS4: A481D) and blue (NS1: D531G). (B) CHIKV genome scheme exhibiting all mutations reconstructed and associated with RJ clades. Non-synonymous mutations are numbered from 1 to 18 and their positions and corresponding amino acid replacements are indicated by black vertical lines. Similarly, vertical gray lines indicate the position of the synonymous mutations inferred. Non-structural and structural open reading frames are colored in dark green and light green, respectively. (TIFF)</p

Fig 2 -

Symptoms distribution, RT-qPCR Ct values dynamics and their correlation with genome sequencing coverage. (A) Distribution of symptoms exhibited by all CHIKV positive patients in the Duque de Caxias cohort. (B) The time lag between symptoms onset and sample collection dates exhibits correlation with RT-qPCR Ct values. As infection proceeds, viral loads decrease (Cts increase) likely due to immunological response. (C) Negative correlation between RT-qPCR Cts and genome sequencing coverage. Sequences characterized from samples with higher viral load (lower Cts) tend to exhibit higher coverage, although no strong statistical correlation was inferred on a linear model (p = 0.08).</p

<i>R</i>_<i>0</i> estimates and sensitivity analysis.

R0 estimates obtained with epidemiological data under different generation time distributions (gamma distributions with means 10, 14 and 20, and constant standard deviation: 6.4 days). (TIFF)</p

<i>R</i>_<i>t</i> estimates and sensitivity analysis.

(A)Rt estimate performed with different generation time (GT) distributions (gamma distributions with means 10, 14 and 20, and constant standard deviation: 6.4 days). Colors indicate estimates for different GTs (10: gray, 14: yellow, 20: blue). (B) Rt estimates performed with different sliding window lengths (3 to 8 weeks). Colors indicate estimates for different window lengths (3: salmon, 4: yellow, 5: green, 6: blue, 7: purple, 8: pink). Solid lines indicate mean values and ribbons indicate the 95% confidence intervals. The dashed lines denote the critical epidemic threshold (Rt = 1). (TIFF)</p

Sample metadata and sequencing statistics for Duque de Caxias samples.

Sample metadata and sequencing statistics for Duque de Caxias samples.</p

Map of Brazil and Rio de Janeiro state, state-level weekly CHIKV incidence in the 2016–2018 period and <i>R</i>_<i>t</i> estimate.

(A) Map of Brazil with all states colored in light gray, except for Rio de Janeiro, in green. (B) Rio de Janeiro map with municipality borders delimitation. The municipality of Duque de Caxias, where samples for genome sequencing were collected, is colored yellow. (C) State level weekly incidence data. Bars correspond to the number of CHIKV cases. (D) Estimate for Rt between 2016 and 2018 (left y-axis). The solid line indicates mean values, while the ribbon indicates the 95% confidence interval. The dashed line represents index P values, a measure of transmissibility potential for the vector Aedes aegypti (right y-axis). The dotted line marks the critical epidemic threshold (Rt = 1). Maps shapefiles were downloaded from Instituto Brasileiro de Geografia e Estatística (IBGE) at https://www.ibge.gov.br/en/geosciences/territorial-organization/territorial-meshes/18890-municipal-mesh.html (last accessed 26 June 2023).</p

Maximum likelihood phylogenetic analysis of global and ECSA-American datasets.

(A) Phylogenetic tree inferred from the global dataset. All new characterized genomes clustered within the ECSA-Br clade. Names of lineages and relevant clades are indicated. SH-aLRT statistical support values for these clades are shown close to their defining nodes (colored in red). (B) Phylogeny inferred from the filtered ECSA-American dataset. Tip shapes are colored according to sampling location (Centre-West region: purple, North region: yellow, Northeast region: red, Rio de Janeiro state: green). Sequences generated in this study are highlighted with red circles around the tip shapes. Clades composed mostly by RJ sequences are indicated along their SH-aLRT support values. (C) The root-to-tip regression plot, which indicates a strong temporal signal (R2 = 0.72, slope = 5.19 x 10−4). Scale bars represent substitutions per site (s/s).</p