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The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

We characterized 3 autochthonous dengue virus serotype 3 cases and 1 imported case from 2 states in the North and South Regions of Brazil, 15 years after Brazil's last outbreak involving this serotype. We also identified a new Asian lineage recently introduced into the Americas, raising concerns about future outbreaks.

Objectives: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection., Methods: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252)., Results: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations., Conclusions: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating., (© 2020 The Authors Tropical Medicine & International Health Published by John Wiley & Sons Ltd.)

Background: Yellow fever (YF) is a severe, infectious, but non-communicable arboviral hemorrhagic disease. In the last decades, yellow fever virus (YFV) infections have been prevalent in endemic areas in Brazil, affecting human and non-human primate (NHP) populations. Monitoring of NHP infection started in 1999, and reports of epizootic diseases are considered important indicators of viral transmission, particularly in relation to the sylvatic cycle. This study presents the monitoring of YFV by real-time RT-PCR and the epidemiological findings related to the deaths of NHPs in the south-eastern states and in the north-eastern state of Bahia, during the outbreak of YF in Brazil during 2017 and 2018., Methods: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were analyzed by real-time reverse transcription polymerase chain reaction (rtRT-PCR)., Results: A total of 4198 samples from 2099 NHPs from south-eastern and north-eastern Brazilian states were collected between 2017 and 2018. The samples were subjected to molecular diagnostics for YFV detection using real-time reverse transcription polymerase chain reaction (rtRT-PCR) techniques. Epizootics were coincident with human YF cases. Furthermore, our results showed that the YF frequency was higher among marmosets (Callithrix sp.) than in previous reports. Viremia in species of the genus Alouatta and Callithrix differed greatly., Discussion: Our results indicate a need for further investigation of the role of Callithrix spp. in the transmission cycles of YFV in Brazil. In particular, YFV transmission was observed in a region where viral circulation has not been recorded for decades and thus vaccination has not been previously recommended., Conclusions: This highlights the need to straighten epizootic surveillance and evaluate the extent of vaccination programmes in Brazil in previously considered "YFV-free" areas of the country.

The recent reemergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the rapid dissemination of the virus in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil, we generated 18 complete and nearly complete genomes from the peak of the epidemic curve from nonhuman primates (NHPs) and human infected cases across the Espírito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source, and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic, YFV was reintroduced from Minas Gerais to the Espírito Santo and Rio de Janeiro states multiple times between 2016 and 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce the idea that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in understanding arbovirus epidemics. IMPORTANCE Arbovirus infections in Brazil, including yellow fever, dengue, zika, and chikungunya, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from nonhuman primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control., (Copyright © 2019 American Society for Microbiology.)

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Since December 2016, Brazil has experienced an unusually large outbreak of yellow fever (YF). Whether urban transmission may contribute to the extent of the outbreak is unclear. The objective of this study was to characterize YF virus (YFV) genomes and to identify spatial patterns to determine the distribution and origin of YF cases in Minas Gerais, Espírito Santo and Rio de Janeiro, the most affected Brazilian states during the current YFV outbreak., Methods: We characterized near-complete YFV genomes from 14 human cases and two nonhuman primates (NHP), sampled from February to April 2017, retrieved epidemiologic data of cases and used a geographic information system to investigate the geospatial spread of YFV., Results: All YFV strains were closely related. On the basis of signature mutations, we identified two cocirculating YFV clusters. One was restricted to the hinterland of Espírito Santo state, and another formed a coastal cluster encompassing several hundred kilometers. Both clusters comprised strains from humans living in rural areas and NHP. Another NHP lineage clustered in a basal relationship. No signs of adaptation of YFV strains to human hosts were detected., Conclusions: Our data suggest sylvatic transmission during the current outbreak. Additionally, cocirculation of two distinct YFV clades occurring in humans and NHP suggests the existence of multiple sylvatic transmission cycles. Increased detection of YFV might be facilitated by raised awareness for arbovirus-mediated disease after Zika and chikungunya virus outbreaks. Further surveillance is required, as reemergence of YFV from NHPs might continue and facilitate the appearance of urban transmission cycles., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: During 2015-16 Brazil experienced the largest epidemic of Zika virus ever reported. This arthropod-borne virus (arbovirus) has been linked to Guillain-Barré syndrome (GBS) in adults but other neurological associations are uncertain. Chikungunya virus has caused outbreaks in Brazil since 2014 but associated neurological disease has rarely been reported here. We investigated adults with acute neurological disorders for Zika, chikungunya and dengue, another arbovirus circulating in Brazil., Methods: We studied adults who had developed a new neurological condition following suspected Zika virus infection between 1st November 2015 and 1st June 2016. Cerebrospinal fluid (CSF), serum, and urine were tested for evidence of Zika, chikungunya, and dengue viruses., Results: Of 35 patients studied, 22 had evidence of recent arboviral infection. Twelve had positive PCR or IgM for Zika, five of whom also had evidence for chikungunya, three for dengue, and one for all three viruses. Five of them presented with GBS; seven had presentations other than GBS, including meningoencephalitis, myelitis, radiculitis or combinations of these syndromes. Additionally, ten patients positive for chikungunya virus, two of whom also had evidence for dengue virus, presented with a similar range of neurological conditions., Conclusions: Zika virus is associated with a wide range of neurological manifestations, including central nervous system disease. Chikungunya virus appears to have an equally important association with neurological disease in Brazil, and many patients had dual infection. To understand fully the burden of Zika we must look beyond GBS, and also investigate for other co-circulating arboviruses, particularly chikungunya.

Background: Zika virus (ZIKV) has been identified in several body fluids of infected individuals. In most cases, it remained detected in blood from few days to 1 week after the onset of symptoms, and can persist longer in urine and in semen. ZIKV infection can have dramatic consequences such as microcephaly and Guillain-Barré syndrome. ZIKV sexual transmission has been documented. A better understanding of ZIKV presence and persistence across biologic compartments is needed to devise rational measures to prevent its transmission., Methods: This observational cohort study will recruit non-pregnant participants aged 18 years and above with confirmed ZIKV infection [positive reverse transcriptase-polymerase chain reaction (RT-PCR) test in blood and/or urine]: symptomatic men and women in ZIKV infection acute phase, and their symptomatic or asymptomatic household/sexual infected contacts. Specimens of blood, urine, semen, vaginal secretion/menstrual blood, rectal swab, oral fluids, tears, sweat, urine and breast milk (if applicable) will be collected at pre-established intervals and tested for ZIKV RNA presence by RT-PCR, other co-infection (dengue, Chikungunya, HIV, hepatitis B and C, syphilis), antibody response (including immunoglobulins M and G), plaque reduction neutralization test (if simultaneously positive for ZIKV and dengue), and ZIKV culture and RNA sequencing. Data on socio-demographic characteristics and comorbidities will be collected in parallel. Participants will be followed up for 12 months., Discussion: This prolonged longitudinal follow-up of ZIKV infected persons with regular biologic testing and data collection will offer a unique opportunity to investigate the presence and persistence of ZIKV in various biologic compartments, their clinical and immunological correlates as well as the possibility of ZIKV reactivation/reinfection over time. This valuable information will substantially contribute to the body of knowledge on ZIKV infection and serve as a base for the development of more effective recommendation on the prevention of ZIKV transmission., Trial Registration: NCT03106714 . Registration Date: April, 7, 2017.

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

This article discusses the peculiar conditions that favoured the unexpected introduction of Zika virus into the poorest northeastern region of Brazil in 2015, its speed of transmission to other Brazilian states, other Latin American countries and other regions, and the severity of related neurological disorders in newborns and adults. Contrasting with evidence that Zika had so far caused only mild cases in humans in the last six decades, the epidemiological scenario of this outbreak in Brazil indicates dramatic health effects: in 2015, an increase of 20-fold in notified cases of microcephaly and/or central nervous system (CNS) alterations suggestive of Zika congenital infection, followed by an exponential increase in 2016, with 2366 cumulative cases confirmed in the country by the end of December 2016. A significant increase in Guillain-Barré syndrome in adults has also been reported. Factors involved in viral dissemination, neural pathogenesis and routes of transmission in Brazil are examined, such as the role of social and environmental factors and the controversies involved in the hypothesis of antibody-dependent enhancement, to explain the incidence of congenital Zika syndrome in Brazil. Responses to the Zika outbreak and the development of new products are also discussed.

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

Background: In 2015, Brazil was faced with the cocirculation of three arboviruses of major public health importance. The emergence of Zika virus (ZIKV) presents new challenges to both clinicians and public health authorities. Overlapping clinical features between diseases caused by ZIKV, Dengue (DENV) and Chikungunya (CHIKV) and the lack of validated serological assays for ZIKV make accurate diagnosis difficult., Methodology / Principal Findings: The outpatient service for acute febrile illnesses in Fiocruz initiated a syndromic clinical observational study in 2007 to capture unusual presentations of DENV infections. In January 2015, an increase of cases with exanthematic disease was observed. Trained physicians evaluated the patients using a detailed case report form that included clinical assessment and laboratory investigations. The laboratory diagnostic algorithm included assays for detection of ZIKV, CHIKV and DENV. 364 suspected cases of Zika virus disease were identified based on clinical criteria between January and July 2015. Of these, 262 (71.9%) were tested and 119 (45.4%) were confirmed by the detection of ZIKV RNA. All of the samples with sequence information available clustered within the Asian genotype., Conclusions / Significance: This is the first report of a ZIKV outbreak in the state of Rio de Janeiro, based on a large number of suspected (n = 364) and laboratory confirmed cases (n = 119). We were able to demonstrate that ZIKV was circulating in Rio de Janeiro as early as January 2015. The peak of the outbreak was documented in May/June 2015. More than half of the patients reported headache, arthralgia, myalgia, non-purulent conjunctivitis, and lower back pain, consistent with the case definition of suspected ZIKV disease issued by the Pan American Health Organization (PAHO). However, fever, when present, was low-intensity and short-termed. In our opinion, pruritus, the second most common clinical sign presented by the confirmed cases, should be added to the PAHO case definition, while fever could be given less emphasis. The emergence of ZIKV as a new pathogen for Brazil in 2015 underscores the need for clinical vigilance and strong epidemiological and laboratory surveillance.

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.

In human lungs, the earliest encounter of Mycobacterium tuberculosis, the agent of tuberculosis, involves alveolar epithelial cells. Droplets expectorated by a patient with tuberculosis are likely to contain a mixed population of M. tuberculosis cells in different physiologic and metabolic states from the lung lesions of the patient. Here, we compared the chemokine expression patterns of human epithelial cell line A549 and RAW 264.7 macrophage cells infected with wild-type M. tuberculosis H37Rv against patterns induced by a mutant that accumulates free mycolic acids in its cell wall (Δmce1). We also examined the effect of free mycolic acids on toll-like receptor-2 (TLR-2). Wild-type M. tuberculosis induced significantly higher levels of IL-8, MCP-1, RANTES, and IP-10 in both cell types than did Δmce. Free mycolic acids reduced the ability of the mammalian cells to respond to a TLR-2 agonist in a dose-dependent manner. These observations suggest that differences in mycolic acid abundance in the M. tuberculosis cell wall can affect TLR-2-mediated pro-inflammatory response in both epithelial and macrophage cells. The final fate of a new infection may be ultimately determined by the proportion of M. tuberculosis cells expressing free mycolates in the infecting inoculum population., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The live-attenuated yellow fever 17D virus is one of the most outstanding human vaccines ever developed. It induces efficacious immune responses at a low production cost with a well-established manufacture process. These advantages make the YF17D virus attractive as a vector for the development of new vaccines. At the beginning of vector development studies, YF17D was genetically manipulated to express other flavivirus prM and E proteins, components of the viral envelope. While these 17D recombinants are based on the substitution of equivalent YF17D genes, other antigens from unrelated pathogens have also been successfully expressed and delivered by recombinant YF17D viruses employing alternative strategies for genetic manipulation of the YF17D genome. Herein, we discuss these strategies in terms of possibilities of single epitope or larger sequence expression and the main properties of these replication-competent viral platforms.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).