Tiago Gräf

Dengue fever, caused by dengue virus (DENV), is one of the most significant mosquito-borne diseases globally, with increasing risk due to the expansion of Aedes vectors into subtropical and temperate zones. Brazil accounted for 70% of dengue cases reported worldwide in 2024, and the virus has recently become endemic in the southernmost state of Rio Grande do Sul. Here, we integrate entomological, epidemiological and environmental data and generate complementary genomic data to investigate DENV dynamics in Southern Brazil from 2015 to 2023. During this period, Aedes aegypti expanded across state municipalities, with a marked increase in dengue cases from 2020 onward. Nearly half of the municipalities surpassed the incidence threshold of ≥20 cases per 100,000 residents per year by 2023, indicating endemic transmission. Genomic analysis of 665 new DENV genomes is consistent with the local persistence of viral lineages across years, driving seasonal outbreaks. Moreover, Northwest and Metropolitan regions are identified as key hubs for viral spread within the Rio Grande do Sul state. These findings highlight the north-to-south expansion of dengue into temperate regions, emphasizing the urgent need for intensification of vector control and surveillance in newly endemic areas to mitigate dengue burden in a mostly naive human population.

Rocio virus (ROCV), a neurotropic arbovirus of the Orthoflavivirus genus, caused Brazil’s largest encephalitis outbreak in the 1970s but has since been rarely detected. We report the first ROCV encephalitis clinical case after nearly 40 years and isolated and sequenced the virus from CNS tissue of a horse. Metatranscriptomics enabled full-genome sequencing, revealing divergence from the reference sequence at an evolutionary rate of 5.8–7.0 × 10⁻⁴, consistent with Orthoflavivirus . Thirty-one non-synonymous substitutions were identified, with the envelope protein most affected. Findings indicate ROCV cryptic circulation and ongoing evolution. Horses may act as sentinels, underscoring the need for strengthened ROCV genomic surveillance.

Dengue fever, caused by the dengue virus (DENV), is the most important mosquito-borne disease impacting human health globally, it is particularly prevalent in tropical regions where Aedes vectors thrive. However, due to a recent expansion of the main dengue vectors to subtropical and temperate zones in the last decades there is an increasing human population at risk. Brazil is a global hotspot for dengue, accounting for 70% of all dengue cases reported worldwide in 2024. Noteworthy, dengue became endemic even in the southernmost Brazilian state of Rio Grande do Sul, which until recently had only imported cases. In this study, we integrated entomological, epidemiological, and high-resolution genomic data to investigate DENV transmission dynamics in Southern Brazil over nine years. From 2015 to 2023, we detected a partial to full invasion of the state municipalities by Aedes aegypti , with dengue cases increasing substantially from 2020 onwards. The suitability index for transmission effectively predict the intensity of the outbreaks in the region, providing a two-month lead time before the surge in cases, representing an interesting proxy to forecast the outbreak size in the next few years. Invaded municipalities that surpassed a given case incidence level also increased, and by 2023, nearly half of the municipalities of the state surpassed that threshold, suggesting that dengue became endemic in the region. By analyzing 665 newly generated DENV genomes, we observed persistent overwintering lineages driving seasonal outbreaks, with the Northwest and Metropolitan regions serving as the main transmission hubs for dengue spread in the state. These regions were also the first to experience the endemic establishment of dengue lineages, making them key areas for targeted control efforts to mitigate further local outbreaks and the virus spread to other regions. These findings underscore the expansion of DENV-affected areas and increasing outbreaks into temperate zones, likely driven by the spread of the vector, associated with increased infestation level of the vector and widespread viral transmission in an immunologically naive population. In this context, enhanced control efforts are critical to mitigate the dengue burden in previously non-endemic areas.

The Brazilian western Amazon is experiencing its largest laboratory-confirmed Oropouche virus (OROV) outbreak, with more than 6,300 reported cases between 2022 and 2024. Here, we sequenced and analyzed 382 OROV genomes from human samples collected in Amazonas, Acre, Rondônia, and Roraima states, between August 2022 and February 2024, to uncover the origin and genetic evolution of OROV in the current outbreak. Genomic analyses revealed that the upsurge of OROV cases in the Brazilian Amazon coincides with spread of a novel reassortant lineage containing the M segment of viruses detected in the eastern Amazon region (2009-2018) and the L and S segments of viruses detected in Peru, Colombia, and Ecuador (2008-2021). The novel reassortant likely emerged in the Amazonas state between 2010 and 2014 and spread through long-range dispersion events during the second half of the 2010s. Phylodynamics reconstructions showed that the current OROV spread was mainly driven by short-range (< 2 km) movements consistent with the flight range of vectors. Nevertheless, a substantial proportion (22%) of long-range (> 10 km) OROV migrations were also detected, consistent with viral dispersion by humans. Our data provides a view of the unprecedented spread and evolution of OROV in Brazilian western Amazon region.

Dengue virus cases are on the rise globally, and strategies to reduce new infections by controlling its primary vector, the mosquito Aedes aegypti, represent a promising biotechnological approach. However, the interaction between virus serotypes and genotypes with Aedes aegypti is poorly characterized at the molecular level, as well as in terms of life-history traits related to vector capacity and mosquito disease tolerance. Here, we infected Aedes aegypti with two philogenetically distant strains of Dengue virus serotype 4 genotype II: a laboratory-adapted strain, DENV4 - TVP/360, and a recent clinical strain isolated from southern Brazil, DENV4 - LRV 13/422. These strains, which exhibit 26 amino acid differences in their sequences, have been shown in to produce distinct immune phenotypes in vertebrate cells. Here, we assessed various life-history traits of Aedes aegypti, including mortality, fecundity, fertility, and induced flight capacity, as well as vector competence-related parameters such as infection intensity and prevalence, following exposure to different viral concentrations. We found that each viral strain differentially affected mosquito traits. While neither strain significantly reduced mosquito lifespan, Aedes aegypti infection prevalence was influenced by the initial dose of DENV4 - TVP/360. This laboratory-adapted strain also enhanced mosquito induced-flight capacity at early (24 hours post-infection - DPI) and late (21 DPI) time points. On the other hand, the recent clinical isolate, DENV4 - LRV 13/422, specifically reduced Aedes aegypti fertility. A better understanding of how different arbovirus strains influence mosquito life-history traits connected to disease spread will be critical in public health efforts to mitigate arbovirus outbreaks that are focused on the mosquito vector.

Brazil's COVID-19 response has faced challenges due to the continuous emergence of variants of concern (VOCs), emphasizing the need for ongoing genomic surveillance and retrospective analyses of past epidemic waves. Rio Grande do Sul (RS), Brazil's southernmost state, has crucial international borders and trades with Argentina and Uruguay, along with significant domestic connections. The source and sink of transmission with both national and international hubs raises questions about the RS role in the transmission of the virus, which has not been fully explored. Nasopharyngeal samples from various municipalities in RS were collected between June 2020 and July 2022. SARS-CoV-2 whole genome amplification and sequencing were performed using high-throughput Illumina sequencing. Bioinformatics analysis encompassed the development of scripts and tools to take into account epidemiological information to reduce sequencing disparities bias among the regions/countries, genome assembly, and large-scale phylogenetic reconstruction. Here, we sequenced 1,480 SARS-CoV-2 genomes from RS, covering all major regions. Sequences predominantly represented Gamma (April-June 2021) and Omicron (January-July 2022) variants. Phylogenetic analysis revealed a regional pattern for transmission dynamics, particularly with Southeast Brazil for Gamma, and a range of inter-regional connections for Delta and Omicron within the country. On the other hand, international and cross-border transmission with Argentina and Uruguay was rather limited. We evaluated the three VOCs circulation over two years in RS using a new subsampling strategy based on the number of cases in each state during the circulation of each VOC. In summary, the retrospective analysis of genomic surveillance data demonstrated that virus transmission was less intense between country borders than within the country. These findings suggest that while non-pharmacological interventions were effective to mitigate transmission across international land borders in RS, they were unsuficient to contain transmission at the domestic level.

Latin America and Caribbean (LAC) regions were an important epicenter of the COVID-19 pandemic and SARS-CoV-2 evolution. Through the COVID-19 Genomic Surveillance Regional Network (COVIGEN), LAC countries produced an important number of genomic sequencing data that made possible an enhanced SARS-CoV-2 genomic surveillance capacity in the Americas, paving the way for characterization of emerging variants and helping to guide the public health response. In this study we analyzed approximately 300,000 SARS-CoV-2 sequences generated between February 2020 and March 2022 by multiple genomic surveillance efforts in LAC and reconstructed the diffusion patterns of the main variants of concern (VOCs) and of interest (VOIs) possibly originated in the Region. Our phylogenetic analysis revealed that the spread of variants Gamma, Lambda and Mu reflects human mobility patterns due to variations of international air passenger transportation and gradual lifting of social distance measures previously implemented in countries. Our results highlight the potential of genetic data to reconstruct viral spread and unveil preferential routes of viral migrations that are shaped by human mobility patterns.