Cryptic circulation of chikungunya virus in São Jose do Rio Preto, Brazil, 2015-2019

Abstract

Background: Chikungunya virus (CHIKV) has spread across Brazil with varying incidence rates depending on the affected areas. Due to cocirculation of arboviruses and overlapping disease symptoms, CHIKV infection may be underdiagnosed. To understand the lack of CHIKV epidemics in São José do Rio Preto (SJdRP), São Paulo (SP), Brazil, we evaluated viral circulation by investigating anti-CHIKV IgG seroconversion in a prospective study of asymptomatic individuals and detecting anti-CHIKV IgM in individuals suspected of dengue infection, as well as CHIKV presence in Aedes mosquitoes. The opportunity to assess two different groups (symptomatic and asymptomatic) exposed at the same geographic region aimed to broaden the possibility of identifying the viral circulation, which had been previously considered absent.

Methodology/principal findings: Based on a prospective population study model and demographic characteristics (sex and age), we analyzed the anti-CHIKV IgG seroconversion rate in 341 subjects by ELISA over four years. The seroprevalence increased from 0.35% in the first year to 2.3% after 3 years of follow-up. Additionally, we investigated 497 samples from a blood panel collected from dengue-suspected individuals during the 2019 dengue outbreak in SJdRP. In total, 4.4% were positive for anti-CHIKV IgM, and 8.6% were positive for IgG. To exclude alphavirus cross-reactivity, we evaluated the presence of anti-Mayaro virus (MAYV) IgG by ELISA, and the positivity rate was 0.3% in the population study and 0.8% in the blood panel samples. In CHIKV and MAYV plaque reduction neutralization tests (PRNTs), the positivity rate for CHIKV-neutralizing antibodies in these ELISA-positive samples was 46.7%, while no MAYV-neutralizing antibodies were detected. Genomic sequencing and phylogenetic analysis revealed CHIKV genotype ECSA in São José do Rio Preto, SP. Finally, mosquitoes collected to complement human surveillance revealed CHIKV positivity of 2.76% of A. aegypti and 9.09% of A. albopictus (although it was far less abundant than A. aegypti) by RT-qPCR.

Conclusions/significance: Our data suggest cryptic CHIKV circulation in SJdRP detected by continual active surveillance. These low levels, but increasing, of viral circulation highlight the possibility of CHIKV outbreaks, as there is a large naïve population. Improved knowledge of the epidemiological situation might aid in outbreaks prevention.

Fig 1. Area of Study.

(A) Geopolitical map of South America, showing Brazil (dark green), and São Paulo state (with the latter highlighted in green, São José do Rio Preto (black point), (B) São José do Rio Preto located in the northeastern region of São Paulo State, and (C) the Vila Toninho neighborhood located in the Southeastern part of the city. Shapefile downloaded from https://portaldemapas.ibge.gov.br/portal.php#homepage.

Fig 2. CHIKV antibodies of participants in the study in the Vila Toninho neighborhood.

Subtitle: Flowchart showing the presence of chikungunya antibodies (Anti-CHIKV IgG) in participants in the study over four years, during the collection period that occurred between October and March of 2015–2019 (Entry Baseline 2015/2016, Follow-up A01 2016/2017, Follow-up A02 2017/2018 and Follow-up A03 2018–2019, as demonstrated in each line of the flowchart) in the Vila Toninho neighborhood. The participants presenting chikungunya-positive antibodies (CHIKV +) are highlighted in pink, while those without chikungunya antibodies (CHIKV -) are shown in blue. CHIKV +: positive samples; *CHIKV +/-: equivocal samples; and CHIKV -: negative samples, as determined by anti-CHIKV IgG ELISA.

Fig 3. Spatial Distribution of Samples from Population Study According to Anti-CHIKV IgG and PRNT Assays.

Thematic map showing the spatial distribution of positive (orange) and borderline positive (dark blue) serological results, according to anti-CHIKV IgG ELISA (A), and positive (red) or negative (blue) seroconversion samples from the population study, according to PRNT assays (B). Shapefile downloaded from https://portaldemapas.ibge.gov.br/portal.php#homepage.

Fig 4. Spatial distribution of A. albopictus mosquitoes positive for CHIKV according to qRT–PCR in Vila Toninho, SJdRP-SP.

Spatial distribution of Ae. aegypti and Ae. albopictus mosquitoes positive for CHIKV as detected by qRT–PCR (Ae. aegypti female and Aedes albopictus (purple), Ae. aegypti male (blue), Ae. aegypti female (yellow) and Ae. albopictus female (pink)) in this study, Vila Toninho, SJdRP-SP. Shapefile downloaded from https://portaldemapas.ibge.gov.br/portal.php#homepage.

Fig 5

Spatial Distribution of Samples According to Serological Status for CHIKV (A) and MAYV (B) by PRNT assay in 2019 during the dengue outbreak in São José do Rio Preto, SP. Seroconversion results of patient samples in 2019 during the dengue outbreak in São José do Rio Preto, SP, illustrating the spatial distribution of serologic status by PRNT₉₀ assay, which was positive (red) and negative (blue) for CHIKV (A) and MAYV (B) and positive (purple) for CHIKV/MAYV cross-reaction and positive (green) for MAYV (C). Shapefile downloaded from https://www.riopreto.sp.gov.br/mapas-rio-preto/.

Fig 6. Maximum likelihood tree of chikungunya virus based on partial envelope gene sequence.

Phylogenetic tree reconstructed using the Maximum-likelihood method with TIM2e+4 as nucleotide substitution model, using Ultrafast Bootstrap (UFBoot) combined with SH-like Approximate Likelihood-ratio test (SH-aLRT). The analysis involved 172 nucleotide (nt) sequences (1,885 nt). Branch lengths are drawn to a scale of nucleotide substitutions per site according to the scale. The analysis was conducted in IQ-TREE v. 2.0.3, and the final tree was visualized and edited in iTOL v. 6.6. The sequence from this study (1362|Brazil|2020) is highlighted in purple within the East/Central/South African genotype clade.