Background From the end of 2016 until the beginning of 2019, Brazil faced a massive sylvatic yellow fever (YF) outbreak. The 2016-2019 YF epidemics affected densely populated areas, especially the Southeast region, causing thousands of deaths of humans and non-human primates (NHP). Methodology/Principal findings We conducted a molecular investigation of yellow fever virus (YFV) RNA in 781 NHP carcasses collected in the urban, urban-rural interface, and rural areas of Minas Gerais state, from January 2017 to December 2018. Samples were analyzed according to the period of sampling, NHP genera, sampling areas, and sampling areas/NHP genera to compare the proportions of YFV-positive carcasses and the estimated YFV genomic loads. YFV infection was confirmed in 38.1% of NHP carcasses (including specimens of the genera Alouatta, Callicebus, Callithrix, and Sapajus), from the urban, urban-rural interface, and rural areas. YFV RNA detection was positively associated with epidemic periods (especially from December to March) and the rural environment. Higher median viral genomic loads (one million times) were estimated in carcasses collected in rural areas compared to urban ones.

The yellow fever (YF) vaccine consists of an attenuated virus, and despite its relative safety, some adverse events following YF vaccination have been described. At the end of 2016, Brazil experienced the most massive sylvatic yellow fever outbreak over the last 70 years and an intense campaign of YF vaccination occurred in Minas Gerais state in Southeast Brazil from 2016 to 2018. The present study aimed to develop a genotyping tool and investigate 21 cases of suspected adverse events following YF vaccination. Initial in silico analyses were performed using partial NS5 nucleotide sequences to verify the discriminatory potential between wild-type and vaccine viruses. Samples from patients were screened for the presence of the YFV RNA, using 5′UTR as the target, and then used for amplification of partial NS5 gene amplification, sequencing, and phylogenetic analysis. Genotyping indicated that 17 suspected cases were infected by the wild-type yellow fever virus, but four cases remained inconclusive. The genotyping tool was efficient in distinguishing the vaccine from wild-type virus, and it has the potential to be used for the differentiation of all yellow fever virus genotypes.

Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Following these massive YF waves, we screened 187 free-living non-human primate (NHPs) carcasses collected throughout the state between January 2019 and June 2021 for YF virus (YFV) using qPCR. One sample belonging to a Callithrix, collected in June 2020, was positive for YFV. The viral strain belonged to the same lineage associated with 2017–2018 outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data from 781 NHPs carcasses collected in 2017–18, we used generalized additive mixed models (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and intensity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained 65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in the eastern part of Minas Gerais state, where 2017–2018 outbreaks affecting humans and NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from urban areas than from urban-rural or rural areas, while infection intensity was significantly lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV infection and intensity were higher during the warm/rainy season compared to the cold/dry season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher exposure to vectors and/or higher virus titers in vectors during this time resulting in the delivery of a higher virus dose and higher viral replication levels within NHPs. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons.

Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Following these massive YF waves, we screened 187 free-living non-human primate (NHPs) carcasses collected throughout the state between January 2019 and June 2021 for YF virus (YFV) using qPCR. One sample belonging to a Callithrix, collected in June 2020, was positive for YFV. The viral strain belonged to the same lineage associated with 2017-2018 outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data from 781 NHPs carcasses collected in 2017-18, we used generalized additive mixed models (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and intensity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained 65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in the eastern part of Minas Gerais state, where 2017-2018 outbreaks affecting humans and NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from urban areas than from urban-rural or rural areas, while infection intensity was significantly lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV infection and intensity were higher during the warm/rainy season compared to the cold/dry season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher exposure to vectors and/or higher virus titers in vectors during this time resulting in the delivery of a higher virus dose and higher viral replication levels within NHPs. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons.

Yellow fever virus (YFV) is the causative agent of yellow fever (YF), a hemorrhagic and viscerotropic acute disease. Severe YF has been described in approximately 15-25% of YF patients, with 20-50% of severe YF cases being fatal. Here we analyzed cerebrospinal fluid (CSF) samples collected during the YF outbreak in Brazil in 2018, aiming to investigate CNS neuroinvasion in fatal YFV cases. YFV RNA was screened by RT-qPCR targeting the 3’UTR region of the YFV genome in CSF. CSF samples were tested for the presence of anti-YFV IgM and neutralizing antibodies, coupled with routine laboratory examinations. Among the 13 patients studied, we detected anti-YFV IgM in CSF from eight patients and YFV RNA in CSF from five patients. YFV RNA genomic load in CSF samples ranged from 1.75x103 to 5.42x103 RNA copies/mL. We genotyped YFV from three CSF samples that grouped with other YFV samples from the 2018 outbreak in Brazil within the South-American I genotype. Even though descriptions of neurologic manifestations due to wild type YFV (WT-YFV) infection are rare, since the last YF outbreak in Brazil in 2017-2018, a few studies have demonstrated WT-YFV RNA in CSF samples from YF fatal cases. Serological tests indicated the presence of IgM and neutralizing antibodies against YFV in CSF samples from two patients. Although the presence of viral RNA, IgM and neutralizing antibodies in CSF samples could indicate neuroinvasiveness, further studies are needed to better elucidate the role of YFV neuroinvasion and possible impacts in disease pathogenesis.