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Adilson Costa,1,2 Elisangela Samartin Pegas Pereira,1 Elvira Cancio Assumpção,1 Felipe Borba Calixto dos Santos,1 Fernanda Sayuri Ota,1 Margareth de Oliveira Pereira,1 Maria Carolina Fidelis,1 Raquel Fávaro,1 Stephanie Selma Barros Langen,1 Lúcia Helena Favaro de Arruda,1 Eva Nydal Abildgaard3 1Department of Dermatology, Pontifícia Universidade Católica de Campinas, Campinas, São Paulo, Brazil; 2KOLderma Clinical Trials Institute, Campinas, São Paulo, Brazil; 3Pfizer Consumer Healthcare, Nutritional Sciences, Copenhagen, Denmark Background: Skin aging is a natural process that may be aggravated by environmental factors. Topical products are the conventional means to combat aging; however, the use of oral supplements is on the rise to assist in the management of aged skin.Objective: The purpose of this study was to assess the effects and safety of an oral supplement containing (per tablet) marine protein (105 mg), vitamin C (27 mg), grape seed extract (13.75 mg), zinc (2 mg), and tomato extract (14.38 mg) in the improvement of skin aging in men.Methods: This single-center, open-label, quasi-experimental clinical study enrolled 47 male subjects, aged 30–45 years, with phototypes I–IV on the Fitzpatrick scale. Subjects received two tablets of the oral supplement for 180 consecutive days. Each subject served as their own control. Clinical assessments were made by medical personnel and by the subjects, respectively. Objective assessments were carried out through pH measurements, sebumetry, corneometry, ultrasound scanning, skin biopsies, and photographic images.Results: Forty-one subjects (87%) completed the study. Clinical improvements on both investigator- and subject-rated outcomes were found for the following parameters: erythema, hydration, radiance, and overall appearance (P

Chikungunya virus (CHIKV) is mainly transmitted by the invasive mosquito Aedes ( Stegomyia ) aegypti in tropical and subtropical regions worldwide. However, genetic adaptations of the virus to the peri domestic mosquito vector Aedes ( Stegomyia ) albopictus has resulted in enhanced vector competence and associated epidemics and may contribute to further geographic expansion of CHIKV. However, evidence-based data on the relative role of Ae. albopictus in CHIKV transmission dynamics are scarce, especially in regions where Ae. aegypti is the main vector, such as in Brazil. Here, we review the CHIKV genotypes circulating in Brazil, spatial and temporal distribution of Chikungunya cases in Brazil, and susceptibility to infection and transmission (i.e., vector competence) of Ae. albopictus for CHIKV to better understand its relative contribution to the virus transmission dynamics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Resck, Câmara, dos Santos, dos Santos, Alto and Honório.)

Background and Objectives: In Brazil, urban arboviruses, such as dengue virus (DENV), Zika virus (ZIKV) and chikungunya virus (CHIKV), constitute a major public health problem, and due to their endemicity and asymptomatic cases, they pose a potential threat to blood donations. Rio de Janeiro (RJ), Brazil, has been impacted by extensive DENV epidemics over the last 30 years and, after 2015, by CHIKV and ZIKV., Materials and Methods: Urban arboviruses DENV, ZIKV and CHIKV were investigated in blood donations (n = 778) at the State Institute of Hematology, HEMORIO (RJ) from 2019 to 2022 by serological and molecular methods., Results: An overall arbovirus exposure was observed in 26.1% of the blood donations. Anti-DENV IgM was detected in 4.0% of samples and two donations were DENV NS1 positive. Positive anti-CHIKV IgM was observed in 4.7% of the donations. Co-detection of anti-CHIKV IgM and anti-DENV IgM was observed in 1.0% of donors, and CHIKV prevalence was 21.3%. All blood donations tested were negative for the DENV, ZIKV and CHIKV RNA., Conclusion: IgM seroprevalence to the arboviruses analyzed here is an indicator of recent infection in asymptomatic donors, showing that the population of blood donors can be a vehicle for new infections, especially during epidemic periods., (© 2024 International Society of Blood Transfusion.)

Oropouche and Mayaro viruses are enzootic arboviruses of public health concern throughout Latin America. Recent outbreaks of OROV in northern region and sporadic autochthonous cases in western region of Brazil, suggest a silent circulation of these neglected viruses. Aiming to investigate the exposure of different species of domestic animals to MAYV and OROV in urban and peri-urban areas of West-Central Brazil, we performed a cross-sectional serosurvey by plaque reduction neutralization test (PRNT). Our findings included neutralizing antibodies for both arboviruses in cattle, dogs and horses, suggesting eventual role of domestic animals in enzootic arbovirus surveillance in Brazil., (© 2024. The Author(s).)

Oropouche orthobunyavirus (OROV) is an arbovirus transmitted by midges that has been involved in outbreaks throughout Central and South America. In Brazil, human cases have been historically concentrated in the northern region of the country. Oropouche fever in humans range from mild clinical signs to rare neurological events, and is considered a neglected tropical disease in Brazil. Due to the clinical similarities to other arboviruses, such as chikungunya and dengue viruses, OROV infections are likely to be underreported. Chikungunya virus (CHIKV) cases in Brazil were first recognized in 2014 in the states of Amapá and Bahia in the north and northeast regions, respectively. Both OROV and CHIKV cause nonspecific symptoms, making clinical diagnosis difficult in a scenario of arbovirus cocirculation. Aiming to investigate OROV transmission during the CHIKV introduction in the state of Amapá located in the Brazilian Amazon, we conducted a retrospective molecular (RT-qPCR) and serological investigation in febrile cases (N = 166) collected between August 2014 and May 2015. All acute serum samples were negative for OROV RNA using RT-qPCR. However, neutralizing antibodies for OROV were detected using a plaque reduction neutralization test (PRNT 90 ) in 10.24% (17/166) of the patients, with neutralizing antibody titers ranging from 20 to ≥640, suggesting the previous exposure of patients to OROV. Regarding CHIKV, recent exposure was confirmed by the detection of CHIKV RNA in 20.25% (33/163) of the patients and by the detection of anti-CHIKV IgM in 28.57% (44/154) of the patients. The additional detection of anti-CHIKV IgG in 12.58% (19/151) of the febrile patients suggests that some individuals had been previously exposed to CHIKV. Whether the OROV exposure reported here occurred prior or during the CHIKV circulation in Amapá, is unknown, but because those arboviral infections share similar clinical signs and symptoms, a silent circulation of enzootic arboviruses during the introduction of exotic arboviruses may occur, and highlights the importance of syndromic cases' surveillance to arboviruses in Brazil.

Redox flow batteries (RFBs) are increasingly being considered for a wide range of energy storage applications, and such devices rely on proton exchange membranes (PEMs) to function. PEMs are high-cost, petroleum-derived polymers that often possess limited durability, variable electrochemical performance, and are linked to discharge of perfluorinated compounds. Alternative PEMs that utilize biobased materials, including lignin and sulfonated lignin (SL), low-cost byproducts of the wood pulping process, have struggled to balance electrochemical performance with dimensional stability. Herein, SL nanoparticles are demonstrated for use as a nature-derived, ion-conducting PEM material. SL nanoparticles (NanoSLs) can be synthesized for increased surface area, uniformity, and miscibility compared with macrosized lignin, improving proton conductivity. After addition of polyvinyl alcohol (PVOH) as a structural backbone, membranes with the highest NanoSL concentration demonstrated an ion exchange capacity of 1.26 meq g -1 , above that of the commercial PEM Nafion 112 (0.98 meq g -1 ), along with a conductivity of 80.4 mS cm -1 in situ, above that of many biocomposite PEMs, and a coulombic efficiency (CE), energy efficiency (EE) and voltage efficiency (VE) of 91%, 68% and 78%, respectively at 20 mA cm -2 . These nanocomposite PEMs demonstrate the potential for valorization of forest biomass waste streams for high value clean energy applications., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Following the first report of zika virus in March 2015, Brazil experienced its largest sylvatic yellow fever outbreak between 2016 and 2019. This study aimed to investigate the circulation of yellow fever virus (YFV) in non-human primates (NHPs) and mosquitoes collected in urban parks and other metropolitan areas of midwest Brazil between 2017 and 2018. Whole blood samples from 80 NHPs, including 48 black-tailed marmosets ( Mico melanurus ) and 2332 mosquitoes from six different genera, were collected in the states of Mato Grosso (MT) and Mato Grosso do Sul (MS) and then tested for YFV by RT-qPCR. Additionally, 23 plasma samples of NHPs were tested for neutralizing antibodies for YFV by a plaque reduction neutralization test (PRNT). No YFV RNA or neutralizing antibodies for YFV were detected in NHPs and mosquitoes from MT and MS. The continuous monitoring of YFV circulation in different species of NHPs and vectors in urban areas is instrumental to quickly assess potentially unknown maintenance cycles of yellow fever at the human-animal interface in Brazil.

Uncaria tomentosa (UT) is a medicinal plant popularly known as cat's claw belonging to the Rubiaceae family that has been reported to display antiviral and anti-inflammatory activities. The chikungunya virus (CHIKV) outbreaks constitute a Brazilian public health concern. CHIKV infection develops an abrupt onset of fever, usually accompanied by a skin rash, besides incapacitating polyarthralgia. There is no vaccine available or treatment for CHIKV infection. The present study evaluates the hydroalcoholic extract of UT bark as a potential antiviral against CHIKV. The in vitro antiviral activity of the UT extract against the Brazilian CHIKV strain was assessed using quantitative reverse transcription polymerase chain reaction, flow cytometry, and plaque assay. Results obtained demonstrated that UT inhibits CHIKV infection in a dose-dependent manner. At the non-cytotoxic concentration of 100 µg/mL, UT exhibited antiviral activity above 90% as determined by plaque reduction assay, and it reduced the viral cytopathic effect. Similarly, a significant virucidal effect of 100 µg/mL UT was observed after 24 and 48 h post-infection. This is the first report on the antiviral activity of UT against CHIKV infection, and the data presented here suggests UT as a potential antiviral to treat CHIKV infection.

Background: Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection., Objectives: The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice., Methods: In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis., Findings: The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm., Main Conclusions: Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Background: Dengue virus can affect the cardiovascular system and men may be at higher risk of severe complications than women. We hypothesized that clinical dengue virus (DENV) infection could induce myocardial alterations of the left ventricle (LV) and that these changes could be detected by transthoracic echocardiography., Methodology/principal Findings: We examined individuals from Acre in the Amazon Basin of Brazil in 2020 as part of the Malaria Heart Study. By questionnaires we collected information on self-reported prior dengue infection. All individuals underwent transthoracic echocardiography, analysis of left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS). We included 521 persons (mean age 40±15 years, 39% men, 50% urban areas) of which 253 (49%) had a history of dengue infection. In multivariable models adjusted for clinical and sociodemographic data, a history of self-reported dengue was significantly associated with lower LVEF (β = -2.37, P < 0.01) and lower GLS (β = 1.08, P < 0.01) in men, whereas no significant associations were found in women (P > 0.05). In line with these findings, men with a history of dengue had higher rates of LV systolic dysfunction (LVEF < 50% = 20%; GLS < 16% = 17%) than those without a history of dengue (LVEF < 50% = 7%; GLS < 16% = 8%; P < 0.01 and 0.06, respectively)., Conclusions/significance: The findings of this study suggest that a clinical infection by dengue virus could induce myocardial alterations, mainly in men and in the LV, which could be detected by conventional transthoracic echocardiography. Hence, these results highlight a potential role of echocardiography for screening LV dysfunction in participants with a history of dengue infection. Further larger studies are warranted to validate the findings of this study., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Kaagaard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mayaro virus (MAYV, Togaviridae) and Oropouche orthobunyavirus (OROV, Peribunyaviridae) are emerging enzootic arboviruses in Latin America. Outbreaks of febrile illness associated with MAYV and OROV have been reported among humans mainly in the northern region of Brazil since the 1980s, and recent data suggest these viruses have circulated also in more populated areas of western Brazil. MAYV shares mosquito vectors with yellow fever virus and it has been historically detected during yellow fever epidemics. Aiming to investigate the transmission of OROV and MAYV at the human-animal interface during a yellow fever, chikungunya and Zika outbreaks in Brazil, we conducted a retrospective molecular investigation in 810 wild and domestic animals, 106 febrile patients, and 22.931 vectors collected from 2016 to 2018 in Cuiaba and Campo Grande metropolitan regions, western Brazil. All samples tested negative for OROV and MAYV RNA by RT-qPCR. Findings presented here suggest no active circulation of MAYV and OROV in the sampled hosts. Active surveillance and retrospective investigations are instrumental approaches for the detection of cryptic and subclinical activity of enzootic arboviruses and together serve as a warning system to implement appropriate actions to prevent outbreaks., Competing Interests: The authors declare no conflict of interest., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Dozens of orthobunyaviruses have been isolated in Brazil, and at least thirteen have been associated with human disease. The Oropouche virus has received most attention for having caused explosive epidemics with hundreds of thousands of cases in the north region between the 1960sand the 1980s, and since then has been sporadically detected elsewhere in the country. Despite their importance, little is known about their enzootic cycles of transmission, amplifying hosts and vectors, and biotic and abiotic factors involved in spillover events to humans. This overview aims to combine available data of neglected orthobunyaviruses of several serogroups, namely, Anopheles A, Anopheles B, Bunyamwera, California, Capim, Gamboa, Group C, Guama, Simbu and Turlock, in order to evaluate the current knowledge and identify research gaps in their natural transmission cycles in Brazil to ultimately point to the future direction in which orthobunyavirus research should be guided.

Chikungunya virus (CHIKV) infection causes intense cytokine/chemokine inflammatory responses and debilitating joint pain. Indoleamine2,3-dioxygenase 1 (IDO-1) is an enzyme that initiates the tryptophan degradation that is important in initial host innate immune defense against infectious pathogens. Besides that, IDO-1 activation acts as a regulatory mechanism to prevent overactive host immune responses. In this study, we evaluated IDO-1 activity and cytokine/chemokine patterns in CHIKV patients. Higher IDO-1 (Kyn/Trp ratio) activation was observed during the early acute phase of CHIKV infection and declined in the chronic phase. Importantly, increased concentrations of Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interferon γ (IFN-γ), C-C motif chemokine ligand 2/Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and C-X-C motif chemokine ligand 10/Interferon Protein-10 (CXCL10/IP-10) were found in the acute phase of infection, while C-C motif chemokine ligand 4/Macrophage Inflammatory Protein 1 β (CCL4/MIP-1β) was found at increased concentrations in the chronic phase. Likewise, CHIKV patients with arthritis had significantly higher concentrations of CCL4/MIP-1β compared to patients without arthritis. Taken together, these data demonstrated increased IDO-1 activity, possibly exerting both antiviral effects and regulating exacerbated inflammatory responses. CCL4/MIP-1β may have an important role in the persistent inflammation and arthritic symptoms following chikungunya infection.

The co-circulation of chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV) in Rio de Janeiro (RJ), Brazil, caused a challenging triple epidemic, as they share similar clinical signs and symptoms and geographical distribution. Here, we aimed to investigate the clinical and laboratorial aspects of chikungunya suspected cases assisted in RJ during the 2018 outbreak, focusing on the differential diagnosis with dengue and zika. All suspected cases were submitted to molecular and/or serological differential diagnostic approaches to arboviruses. A total of 242 cases suspected of arbovirus infection were investigated and 73.6% (178/242) were molecular and/or serologically confirmed as chikungunya. In RT-qPCR confirmed cases, cycle threshold (Ct) values ranged from 15.46 to 35.13, with acute cases presenting lower values. Chikungunya cases were mainly in females (64%) and the most frequently affected age group was adults between 46 to 59 years old (27%). Polyarthralgia affected 89% of patients, especially in hands and feet. No dengue virus (DENV) and Zika virus (ZIKV) infections were confirmed by molecular diagnosis, but 9.5% (23/242) had serological evidence of DENV exposure by the detection of specific anti-DENV IgM or NS1, and 42.7% (76/178) of chikungunya positive cases also presented recent DENV exposure reflected by a positive anti-DENV IgM or NS1 result. A significantly higher frequency of arthritis ( p = 0.023) and limb edema ( p < 0.001) was found on patients with CHIKV monoinfection compared to dengue patients and patients exposed to both viruses. Lastly, phylogenetic analysis showed that the chikungunya cases were caused by the ECSA genotype. Despite the triple arboviruses' epidemic in the state of RJ, most patients with fever and arthralgia investigated here were diagnosed as chikungunya cases, and the incidence of CHIKV/DENV co-detection was higher than that reported in other studies.

Background: Chikungunya virus (CHIKV) causes a febrile syndrome with intense and debilitating arthralgia that can persist for several months or years after complete virus clearance. As there is no specific antiviral treatment or vaccine against CHIKV, identification of serological markers that help clinical management of CHIKV patients is urgent. The High Mobility Group Box 1 (HMGB1) protein is secreted to extracellular milieu and triggers an intense inflammatory process by inducing the overexpression of pro-inflammatory cytokines. HMGB1 plays an important role in several virus diseases as well as in rheumatoid arthritis., Objectives: This study focus on the investigation of HMGB1 serum levels in a sera panel from CHIKV-infected patients in an attempt to assess its potential as a biomarker for chikungunya clinical management., Study Design: Eighty CHIKV-positive samples and 32 samples from healthy donors were subjected to a quantitative HMGB1 ELISA assay to assess the HMGB1 circulating levels., Results: HMGB1 levels were significantly higher in CHIKV-positive samples (516.12 ng/mL, SEM ± 48.83 ng/mL) compared to negative control (31.20 ng/mL, SEM ± 3.24 ng/mL, p < 0.0001). Circulating levels of HMGB1 persisted elevated during the whole acute-phase of disease and correlated with virus titer (p < 0.05)., Conclusions: The present study is the first to describe increased serum levels of HMGB1 in CHIKV infection and its positive correlation with virus titer, suggesting its potential use as a biomarker for diagnosis and treatment of chikungunya fever., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Several protocols for genomic amplification using reverse transcription followed by polymerase chain reaction (RT-PCR), important in the identification of the infecting serotype, have been used in the rapid diagnosis of Dengue Virus (DENV) infections. The qualitative protocol described by Lanciotti et al. (J Clin Microbiol 30: 545-551, 1992) suggested by WHO detects the four DENV serotypes simultaneously in one procedure "semi-nested," generating amplified products with specific sizes in base pairs for each serotype and it has been the most used in the past two decades. However, advances in molecular diagnosis have enabled the development of RT-PCR in real time (qRT-PCR) based on the use of dyes and probes (SYBR green and TaqMan), which is performed in a single step and is capable of providing quantitative data. In addition to quantification, the advantages of qRT-PCR over conventional RT-PCR include speed, greater sensitivity and specificity, and low rate of false positives. Several protocols for the diagnosis and/or quantification of DENV have already been described. Non-PCR-based methods such as reverse transcription loop-mediated isothermal amplification have shown high sensitivities and specificities. RT-PCR and qRT-PCR techniques can be performed using serum, plasma, infected cells, mosquitoes, fresh, and paraffin-embedded tissues. However, despite fast and accurate, they are limited to samples collected during the acute phase of infection (up to 7 days after the onset of symptoms) and require specialized equipment and trained staff., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A reliable and specific diagnosis is imperative in viral diagnosis, both for clinical management and surveillance, and to ensure that early treatment and control measures are carried out. The number of days of illness is important to choose the most appropriate method to be used and for the correct interpretation of the results obtained. Specific IgM is elicited after that period, indicating an active infection and usually lasts up to 3 months. However, in DENV secondary infections, IgM levels may be significantly lower or undetectable. After 10-12 days, a lifetime specific IgG is produced. Routinely, the laboratory diagnosis of DENV infections can be performed by viral isolation and/or detection of viral nucleic acid, serological assays for the detection of specific antibodies (IgM/IgG), antigen (NS1) and the detection of viral antigens in tissues, which are suitable during certain phases of the disease. For serological diagnosis, serum, plasma, or cerebrospinal fluid (CSF) samples may be investigated. If the test is carried out a few days after collection, the specimens can be stored at 4 °C, since the immunoglobulins are stable in serum or plasma. If the storage period is extended, the material must be kept at -20 °C or -70 °C. In serology, several methods can be used to detect specific viral antigens and/or antibodies, produced by the host in response to DENV infection. Routinely, serological tests include the hemagglutination inhibition (HI) assay, the plaque reduction neutralizing test (PRNT), the gold standard assay for dengue immune response characterization, and ELISAs to detect IgM (MAC-ELISA) and IgG (IgG-ELISA)., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Dengue virus (DENV) infection by one of the four serotypes (DENV-1 to 4) may result in a wide spectrum of clinical manifestations, with unpredictable evolution and organ involvement. Due to its association with severe epidemics and clinical manifestations, DENV-2 has been substantially investigated. In fact, the first emergence of a new lineage of the DENV-2 Asian/American genotype in Brazil (Lineage II) in 2008 was associated with severe cases and increased mortality related to organ involvement. A major challenge for dengue pathogenesis studies has been a suitable animal model, but the use of immune-competent mice, although sometimes controversial, has proven to be useful, as histological observations in infected animals reveal tissue alterations consistent to those observed in dengue human cases. Here, we aimed to investigate the outcomes caused by two distinct lineages of the DENV-2 Asian/American genotype in the lung, heart and skeletal muscle tissues of infected BALB/c mice. Tissues were submitted to histopathology, immunohistochemistry, histomorphometry and transmission electron microscopy (TEM) analysis. The viral genome was detected in heart and skeletal muscle samples. The viral antigen was detected in cardiomyocytes and endothelial cells of heart tissue. Heart and lung tissue samples presented morphological alterations comparable to those seen in dengue human cases. Creatine kinase serum levels were higher in mice infected with both lineages of DENV-2. Additionally, statistically significant differences, concerning alveolar septa thickening and heart weight, were observed between BALB/c mice infected with both DENV-2 lineages, which was demonstrated to be an appropriate experimental model for dengue pathogenesis studies on lung, heart and skeletal muscle tissues.

Purpose: This study aimed to analyze the frequency of piroplasmids in the blood of dogs in Rio de Janeiro, compare the performance of microscopic techniques, assess the risk factors associated with infections and also molecularly and morphologically characterize the piroplasmids identified., Methods: In all, 407 blood samples were collected from dogs between 2018 and 2019. These were subjected to microscopic parasitological techniques for thin and thick smears, stained with Giemsa and using a rapid staining kit. The slides were read under an optical microscope and the protozoa were characterized morphometrically. In addition, the blood samples were subjected to molecular characterization for diagnosing piroplasmid species using primers that amplified the gene 18S rRNA., Results: Piroplasmids were detected in 38 (9.3%) samples. Of these, 33 samples presented nucleotide sequences compatible with Babesia vogeli. Most of the positive samples were young, male, defined breeds dogs that had been attended in clinics in São Gonçalo city. Thrombocytopenia and leukopenia were the hematological alterations more observed in positive samples, but positive samples without alterations were also detected. The sex was the only variable that showed statistical differences. Males dogs being more often infected than females (p < 0.05). The microscope slides mostly showed piriform and oval merozoites measuring greater than 2.5 µm in length, which were compatible with B. vogeli. However, smaller forms were also identified, thus demonstrating the polymorphic nature of this parasite., Conclusion: Babesia vogeli was detected in blood samples from dogs in the metropolitan cities of Rio de Janeiro by molecular techniques in different parasite morphotypes., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

The incidence of dengue in Latin America has increased dramatically during the last decade. Understanding the pathogenic mechanisms in dengue is crucial for the identification of biomarkers for the triage of patients. We aimed to characterize the profile of cytokines (IFN-γ, TNF-α, IL-1β, IL-6, IL-18 and IL-10), chemokines (CXCL8/IL-8, CCL2/MCP-1 and CXCL10/IP-10) and coagulation mediators (Fibrinogen, D-dimer, Tissue factor-TF, Tissue factor pathway inhibitor-TFPI and Thrombomodulin) during the dengue-4 epidemic in Brazil. Laboratory-confirmed dengue cases had higher levels of TNF-α ( p < 0.001), IL-6 ( p = 0.005), IL-10 ( p < 0.001), IL-18 ( p = 0.001), CXCL8/IL-8 ( p < 0.001), CCL2/MCP-1 ( p < 0.001), CXCL10/IP-10 ( p = 0.001), fibrinogen ( p = 0.037), D-dimer ( p = 0.01) and TFPI ( p = 0.042) and lower levels of TF ( p = 0.042) compared to healthy controls. A principal component analysis (PCA) distinguished between two profiles of mediators of inflammation and coagulation: protective (TNF-α, IL-1β and CXCL8/IL-8) and pathological (IL-6, TF and TFPI). Lastly, multivariate logistic regression analysis identified high aspartate aminotransferase-to-platelet ratio index (APRI) as independent risk factors associated with severity (adjusted OR: 1.33; 95% CI 1.03-1.71; p = 0.027), the area under the receiver operating characteristics curve (AUC) was 0.775 (95% CI 0.681-0.869) and an optimal cutoff value was 1.4 (sensitivity: 76%; specificity: 79%), so it could be a useful marker for the triage of patients attending primary care centers.

Dengue virus type 2 (DENV-2) is, traditionally, the most studied serotype due to its association with explosive outbreaks and severe cases. In Brazil, almost 20 years after the first introduction in the 1990s, a new lineage (Lineage II) of the DENV-2 Asian/American genotype emerged and caused an epidemic with severe cases and hospitalizations. Severe dengue includes multiple organ failure, and renal involvement can be potentially related to increased mortality. In order to better understand the role of DENV infection in renal injury, here we aimed to investigate the outcomes of infection with two distinct lineages of DENV-2 Asian/American genotype in the kidney of a murine model. BALB/c mice were infected with Lineages I and II and tissues were submitted to histopathology, immunohistochemistry, histomorphometry and ultrastructural analysis. Blood urea nitrogen (BUN) was detected in blood sample accessed by cardiac puncture. A tendency in kidney weight increase was observed in mice infected with both lineages, but urea levels, on average, were increased only in mice infected with Lineage II. The DENV antigen was detected in the tissue of mice infected with Lineage II and morphological changes were similar to those observed in human dengue cases. Furthermore, the parameters such as organ weight, urea levels and morphometric analysis, showed significant differences between the two lineages in the infected BALB/c, which was demonstrated to be a suitable experimental model for dengue pathophysiology studies in kidneys.

Virus-induced infections of the central nervous system (CNS) are among the most serious problems in public health and can be associated with high rates of morbidity and mortality, mainly in low- and middle-income countries, where these manifestations have been neglected. Typically, herpes simplex virus 1 and 2, varicella-zoster, and enterovirus are responsible for a high number of cases in immunocompetent hosts, whereas other herpesviruses (for example, cytomegalovirus) are the most common in immunocompromised individuals. Arboviruses have also been associated with outbreaks with a high burden of neurological disorders, such as the Zika virus epidemic in Brazil. There is a current lack of understanding in Brazil about the most common viruses involved in CNS infections. In this review, we briefly summarize the most recent studies and findings associated with the CNS, in addition to epidemiological data that provide extensive information on the circulation and diversity of the most common neuro-invasive viruses in Brazil. We also highlight important aspects of the prion-associated diseases. This review provides readers with better knowledge of virus-associated CNS infections. A deeper understanding of these infections will support the improvement of the current surveillance strategies to allow the timely monitoring of the emergence/re-emergence of neurotropic viruses.

From 2016 to 2018, Brazil faced the biggest yellow fever (YF) outbreak in the last 80 years, representing a risk of YF reurbanization, especially in megacities. Along with this challenge, the mass administration of the fractionated YF vaccine dose in a naïve population brought another concern: the possibility to increase YF adverse events associated with viscerotropic (YEL-AVD) or neurological disease (YEL-AND). For this reason, we developed a quantitative real time RT-PCR (RT-qPCR) assay based on a duplex TaqMan protocol to distinguish broad-spectrum infections caused by wild-type yellow fever virus (YFV) strain from adverse events following immunization (AEFI) by 17DD strain during the vaccination campaign used to contain this outbreak. A rapid and more accurate RT-qPCR assay to diagnose YFV was established, being able to detect even different YFV genotypes and geographic strains that circulate in Central and South America. Moreover, after testing around 1400 samples from human cases, non-human primates and mosquitoes, we detected just two YEL-AVD cases, confirmed by sequencing, during the massive vaccination in Brazilian Southeast region, showing lower incidence than AEFI as expected.

Dengue (DEN) is the most prevalent arbovirus among humans, and four billion people live at risk of infection. The clinical manifestations of DEN are variable, and the disease may present subclinically or asymptomatically. A quarter of patients develop classical dengue (CD) or severe dengue (SD), which is potentially lethal and involves vascular permeability changes, severe hemorrhage and organ damage. The involvement of the liver is a fairly common feature in DEN, and alterations range from asymptomatic elevation of transaminases to acute liver failure. Since its introduction in Brazil in 1990, two strains of Dengue virus (DENV) serotype 2 (DENV-2) have been detected: Lineage I, which is responsible for an outbreak in 1991, and Lineage II, which caused an epidemic greater than the previous one and had a different epidemiological profile. To date, studies on different strains of the same serotype/genotype and their association with disease severity are scarce. In addition, one of the greatest challenges regarding the study of DEN pathogenesis and the development of drug and vaccine therapies is the absence of an animal model that reproduces the disease as it occurs in humans. The main goals of this study were to assess BALB/c mouse susceptibility experimentally infected by two distinct DENV-2 strains and characterize possible differences in the clinical signs and alterations induced in the liver resulting from those infections. Mice infected by the two DENV-2 lineages gained less weight than uninfected mice; however, their livers were slightly heavier. Increased AST and AST levels were observed in infected mice, and the number of platelets increased in the first 72 h of infection and subsequently decreased. Mice infected with both lineages presented leukocytosis but at different times of infection. The histopathological changes induced by both lineages were similar and comparable to the changes observed in DEN fatal cases. The viral genome was detected in two liver samples. The results demonstrate the susceptibility of BALB/c mice to both DENV-2 lineages and suggest that the changes induced by those strains are similar, although for some parameters, they are manifested at different times of infection.

In Brazil, chikungunya emerged in 2014, and by 2016, co-circulated with other arbovirosis, such as dengue and zika. ELISAs (Enzyme-Linked Immunosorbent Assays) are the most widely used approach for arboviruses diagnosis. However, some limitations include antibody cross reactivities when viruses belong to the same genus, and sensitivity variations in distinct epidemiological scenarios. As chikungunya virus (CHIKV) is an alphavirus, no serological cross reactivity with dengue virus (DENV) should be observed. Here, we evaluated a routinely used chikungunya commercial IgM (Immunoglobulin M) ELISA test (Anti-Chikungunya IgM ELISA, Euroimmun) to assess its performance in confirming chikungunya in a dengue endemic area. Samples ( n = 340) representative of all four DENV serotypes, healthy individuals and controls were tested. The Anti-CHIKV IgM ELISA test had a sensitivity of 100% and a specificity of 25.3% due to the cross reactivities observed with dengue. In dengue acute cases, the chikungunya test showed an overall cross-reactivity of 31.6%, with a higher cross-reactivity with DENV-4. In dengue IgM positive cases, the assay showed a cross-reactivity of 46.7%. Serological diagnosis may be challenging and, despite the results observed here, more evaluations shall be performed. Because distinct arboviruses co-circulate in Brazil, reliable diagnostic tools are essential for disease surveillance and patient management.

The epidemic of coronavirus disease 2019 (COVID-19), caused by a novel Betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a public health emergency worldwide. Few reports indicate that owned pets from households with at least one human resident that was diagnosed with COVID-19 can be infected by SARS-CoV-2. However, the exposure to SARS-CoV-2 of pets from households with no COVID-19 cases or stray animals remains less assessed. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT90), we investigated the infection and previous exposure of dogs and cats to SARS-CoV-2 during the ongoing COVID-19 epidemic in Rio de Janeiro, Brazil. From June to August 2020, 96 animals were sampled, including 49 cats (40 owned and 9 stray) and 47 dogs (42 owned and 5 stray). Regarding owned pets, 75.6% (62/82) belonged to households with no COVID-19 cases. Samples included serum, and rectal and oropharyngeal swabs. All swabs were negative for SARS-CoV-2 RNA, but serum samples of a stray cat and a stray dog presented neutralizing antibodies for SARS-CoV-2, with PRNT90 titer of 80 and 40, respectively. Serological data presented here suggest that not only owned pets from households with COVID19 cases, but also stray animals are being exposed to SARS-CoV-2 during the COVID-19 pandemic., Competing Interests: The authors have declared that no competing interests exist.

Zika and Dengue viruses present considerable immunological cross-reactivity, resulting in a troublesome serodiagnosis due to occurrence of false positive results. Due to Brazil's wide variety of circulating flaviviruses we aimed to access the use of in house serological tests adapted by National Reference Laboratory for Arboviruses in Brazil and evaluate commercial tests available. We evaluated in house IgM ELISAs for the individual detection of anti-ZIKV, -DENV, and -YFV IgM, against a panel of samples positive for dengue, zika, yellow fever, Rocio, Ilheus, Saint Louis encephalitis, West Nile and chikungunya. We also evaluated two commercial kits for dengue and zika IgM detection recommended by the Brazilian Ministry of Health in 2015. The sensitivity and specificity for the in house ZIKV IgM ELISA was 60.0 % and 88.6 % and for the in house DENV IgM ELISA was 100 % and 82.2 %, respectively. The in house YFV IgM ELISA presented 100 % for both sensitivity and specificity. The Novagnost Zika Virus IgM test presented a sensitivity of 47.3 % and specificity of 85.3 % and the Serion ELISA classic Dengue Virus IgM, 92.8 % and 58.9 %, respectively. Overall, both in house ELISAs for ZIKV and DENV adapted and evaluated here, presented better performances than the commercial kits tested., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Plants are widely used in folk medicine because of their pharmacological properties. Ceiba speciosa , popularly known as paineira-rosa or tree-of-wool, is a species found in the Northwest of Rio Grande do Sul, being native of the upper Uruguay River, Brazil. The tea obtained from the stem bark is employed in folk medicine to reduce cholesterol, triacylglycerides, and glucose levels. However, there are no studies in the literature proving its efficacy or the safety of its use. For this study, we used Caenorhabditis elegans as an animal model considering its advantages for risk assessment and pharmacological screenings. For the toxicological tests, C. elegans N2 (wild type) was treated with the aqueous extract of the stem bark of C. speciosa (ECE) at the first larval stage (L1) at concentrations of 5, 25, 50, and 250  μ g/mL. To evaluate biological activities, we challenged the extract for oxidative stress resistance in the presence of paraquat (0.5 mM), H 2 O 2 (1 mM), and against glucose-induced toxicity. Our results demonstrated that ECE did not alter survival rate, pharyngeal pumping, and reproduction of the nematodes. The extract was not able to protect the nematodes against the toxicity induced by prooxidants. Notably, ECE protected against glucotoxicity by increasing worms' life span and by reducing glucose levels. On the other hand, ECE treatment did not reduce lipid accumulation induced by exogenous glucose feeding, as observed in worms which lipid droplets were tagged with GFP. Based on our results, we believe that the extract is indeed promising for further studies focusing on carbohydrates metabolism; however, it needs to be carefully evaluated since the extract does not seem to modulate lipid accumulation., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Fabrine Bianchin dos Santos et al.)

Background: Zika virus (ZIKV) emerged in the Pacific Ocean and subsequently caused a dramatic Pan-American epidemic after its first appearance in the Northeast region of Brazil in 2015. The virus is transmitted by Aedes mosquitoes. We evaluated the role of temperature and infectious doses of ZIKV in vector competence of Brazilian populations of Ae. aegypti and Ae. albopictus., Methodology/principal Findings: Two Ae. aegypti (Rio de Janeiro and Natal) and two Ae. albopictus (Rio de Janeiro and Manaus) populations were orally challenged with five viral doses (102 to 106 PFU / ml) of a ZIKV strain (Asian genotype) isolated in Northeastern Brazil, and incubated for 14 and 21 days in temperatures mimicking the spring-summer (28°C) and winter-autumn (22°C) mean values in Brazil. Detection of viral particles in the body, head and saliva samples was done by plaque assays in cell culture for determining the infection, dissemination and transmission rates, respectively. Compared with 28°C, at 22°C, transmission rates were significantly lower for both Ae. aegypti populations, and Ae. albopictus were not able to transmit the virus. Ae. albopictus showed low transmission rates even when challenged with the highest viral dose, while both Ae. aegypti populations presented higher of infection, dissemination and transmission rates than Ae. albopictus. Ae. aegypti showed higher transmission efficiency when taking virus doses of 105 and 106 PFU/mL following incubation at 28°C; both Ae. aegypti and Ae. albopictus were unable to transmit ZIKV with virus doses of 102 and 103 PFU/mL, regardless the incubation temperature., Conclusions/significance: The ingested viral dose and incubation temperature were significant predictors of the proportion of mosquito's biting becoming infectious. Ae. aegypti and Ae. albopictus have the ability to transmit ZIKV when incubated at 28°C. However Brazilian populations of Ae. aegypti exhibit a much higher transmission potential for ZIKV than Ae. albopictus regardless the combination of infection dose and incubation temperature., Competing Interests: The authors have declared that no competing interests exist.

In Brazil, an epidemic of Zika virus (ZIKV) infections was declared in 2015 that coincided with alarming reports of microcephaly in newborns associated with mother infection. Although the virus has placental tropism, changes in the tissue morphology and immunity of infected patients have not yet been elucidated. Here, we investigated the histopathological and ultrastructural changes along with the immunological profile and the BDNF expression in rare placental material. Tissues were obtained in the 2015-2016 Brazilian epidemic, of ten ZIKV-infected patients during pregnancy, five resulting in cases of fetal microcephaly and five non-microcephaly, compared to five non-infected control placentae. Viral antigens were only detected in samples from the ZIKV infected patients. Infected placentae presented histopathological severe damage, while the ultrastructural evaluation showed abnormal organelles, such as clusters of virus-like particles consistent with the ZIKV dimensions. Increased infiltration of CD68 + and TCD8 + cells, expression of MMPs, cytokines (IFN-γ and TNF-α) and other immunological mediators (RANTES/CCL5 and VEGFR-2) confirmed excessive inflammation and vascular permeability dysfunction. An evaluation of BDNF showed a decrease that could modulate neuronal damage in the developing fetus. The placental changes caused by ZIKV are not pathognomonic, however, the data provide evidence that this infection leads to severe placental injury., (Copyright © 2020 Rabelo, de Souza, Salomão, Machado, Pereira, Portari, Basílio-de-Oliveira, dos Santos, Neves, Morgade, Provance, Higa, Tanuri, de Carvalho and Paes.)

Background: Arboviruses co-circulating within a population that are transmitted by the same vector have the potential to cause coinfections. Coinfections with dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) have been occurring in Brazil, but it is not well-understood how human responses vary during mono- or coinfections and whether they play different roles in pathogenesis., Methods: We investigated the clinical, virological, and immunological status during patients' acute infections, focusing on the CCL/CXC chemokines, proinflammatory, as well as anti-inflammatory cytokines levels quantified by ELISAs. Viral load was determined by qRT-PCR in serum samples from 116 acute DENV, ZIKV, CHIKV, DENV/ZIKV, and CHIKV/ZIKV-infected adult patients from Brazil., Results: Most of the acute patients displayed fever, headache, prostration, and myalgia, regardless of the type of arbovirus infection. Zika viral load was higher in CHIKV/ZIKV coinfected patients compared with ZIKV or DENV/ZIKV infections. All infected individuals presented increased concentrations of C-X-C motif chemokine ligand 10/interferon protein-10 (CXCL10/IP-10), C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and tumor necrosis factor alpha (TNF-α) compared to healthy donors. Interestingly, the ZIKV group separated from CHIKV/ZIKV due to higher levels of interleukin-10 (IL-10) and lower levels of TNF-α. While DENV/ZIKV differentiated from CHIKV due to their higher levels of CCL2/MCP-1, in CHIKV- and CHIKV/ZIKV-infected patients, levels of CXC10/IP-10, CCL2/MCP-1, and migration inhibitory factor (MIF) were associated with CHIKV viral load. By contrast, in DENV/ZIKV- and CHIKV/ZIKV-infected patients, levels of CXCL10/IP-10, CCL2/MCP-1, and TNF-α showed a significant inverse correlation with ZIKV viral load., Conclusions: From all the circulating mediators measured, we detected differences of IL-10, TNF-α, and CCL2/MCP-1 between arbovirus groups. We hypothesize that CXC10/IP-10, CCL2/MCP-1, and MIF in the CHIKV-infected group could regulate the CHIKV viral load, while CXC10/IP-10, CCL2/MCP-1, and TNF-α in DENV/ZIKV, and CHIKV/ZIKV groups, could regulate ZIKV viral load., (© 2020 S. Karger AG, Basel.)

The presence of dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) in Brazil, may result in a difficult diagnosis due to the signs and symptoms shared by those. Moreover, as DENV and ZIKV belong to the same family, serological assays may show a high rate of cross-reactivity. Here, we evaluated a Dengue NS1 capture assay for early and differential diagnosis of dengue during the Zika epidemic occurred in Brazil in 2016. Samples (n = 227) from 218 patients included sera, plasma and urine from previously confirmed acute cases of Zika, dengue and Zika/dengue co-infections. Nine of those patients presented two specimens. The Dengue NS1 test was very specific for dengue diagnosis (99.32%), even in the co-circulation with ZIKV, and exhibited a high accuracy in not detecting acute Zika infections (92.43%). Our findings showed that the dengue NS1 capture test analyzed here was not able to recognize the ZIKV NS1 and its potential for cross-reaction.

Dengue virus (DENV) infections may result in asymptomatic cases or evolve into a severe disease, which involves multiple organ failure. Renal involvement in dengue can be potentially related to an increased mortality. Aiming to better understand the role of DENV in renal injury observed in human fatal cases, post-mortem investigations were performed in four DENV-4 renal autopsies during dengue epidemics in Brazil. Tissues were submitted to histopathology, immunohistochemistry, viral quantification, and characterization of cytokines and inflammatory mediators. Probably due the high viral load, several lesions were observed in the renal tissue, such as diffuse mononuclear infiltration around the glomerulus in the cortical region and in the medullary vessels, hyalinosis arteriolar, lymphocytic infiltrate, increased capsular fibrosis, proximal convoluted tubule (PCT) damage, edema, PCT debris formation, and thickening of the basal vessel membrane. These changes were associated with DENV-4 infection, as confirmed by the presence of DENV-specific NS3 protein, indicative of viral replication. The exacerbated presence of mononuclear cells at several renal tissue sites culminated in the secretion of proinflammatory cytokines and chemokines. Moreover, it can be suggested that the renal tissue injury observed here may have been due to the combination of both high viral load and exacerbated host immune response.

Background: Arboviruses (Zika, dengue and chikungunya) represent a major risk for pregnant women, especially because their vertical transmission can lead to neurological damage in newborns. Early diagnosis can be difficult due to similar clinical presentation with other congenital infections that are associated with congenital abnormalities., Objectives: To investigate the circulation of arboviruses and other pathogens responsible for congenital infections, reporting clinical aspects and geographic distribution of maternal rash in a metropolitan region of Rio de Janeiro (Brazil)., Methods: Cross-sectional study with pregnant women presenting rash attended at the Exanthematic Diseases Unit (Niterói, Rio de Janeiro) from 2015 to 2018. Diagnosis of arboviruses was performed by real-time PCR (RT-qPCR) and laboratorial screening for syphilis, toxoplasmosis, rubella, cytomegalovirus and HIV was assessed. Demographic data was used for georeferencing analysis., Findings: We included 121 pregnant women, of whom Zika virus was detected in 45 cases (37.2%), chikungunya in 33 (27.3%) and dengue in one (0.8%). Five patients presented syphilis, and we observed one case each of listeria, cytomegalovirus, and a syphilis-toxoplasmosis case. Similarity of clinical symptoms was observed in all groups; however, 84.8% of patients with chikungunya presented arthralgia. Following the decline of Zika cases, chikungunya infection was mostly observed during 2017-2018. Considering pregnant women infected with arboviruses and other infections, 41% resided in urban slums, mostly in Niterói., Main Conclusions: Simultaneous circulation of arboviruses and other agents responsible for congenital infections were observed; however, we did not identify co-infections between arboviruses. In this scenario, we emphasize the importance of adequate prenatal care to provide an accurate diagnosis of maternal rash., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Over the last 30 years, extensive dengue epidemics have occurred in Brazil, characterized by emergences and re-emergences of different serotypes, a change in the epidemiological profile and an increase in the number of severe and fatal cases. Here, we present a review on the dengue fatal cases that occurred in Brazil in 30 years (1986-2015)., Methods: We performed an ecological study by using secondary data on dengue fatal cases obtained in the National System of Reported Diseases (Sistema de Informação de Agravos de Notificação -SINAN) and in the Mortality Information System (SIM), both maintained by the Brazilian Ministry of Health. Cases were analyzed by region, demographic variables, clinical classification and complications based on the data available., Results: In 30 years (1986-2015), the Southeast region reported 43% (n = 2225) of all dengue deaths in the country. The Midwest region was responsible for 18% of the fatal cases. After 2000, deaths occurred in almost all states, with the exception of Santa Catarina and Rio Grande do Sul, South region. From 2006 to 2010, the number of deaths increased, with higher rates of mortality, especially in Goiás and Mato Grosso. From 2011 to 2015, Goiás became the state with the highest mortality rate in the country, and Rio Grande do Sul reported its first dengue deaths. In 30 years, a total of 2682 dengue deaths occurred in males and 2455 in females, and an equal distribution between the sexes was observed. From 1986 to 2006, dengue deaths occurred predominantly in individuals over 15 years old, but this scenario changed in 2007-2008. After 2009, fatal cases on individuals above 15 years old became more frequent, with peaks in the years of 2010, 2013 and 2015., Conclusions: The Brazil is experiencing a hyperendemic scenario, which has resulted in the co-circulation of the four DENV serotypes and with the increasing occurrence of severe and fatal cases. The disease surveillance and studies characterizing what has been reported overtime, are still important tools to better understand the factors involved in the disease outcome.

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP + -reduction., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The increase in urbanization and industrialization has contributed to the contamination of different environments by means of xenobiotic compounds, such as heavy metals, causing changes in microbial communities. Among these metals, the Mercury (Hg 2+ ) is one the most prevalent toxic metals for the environment The present study aimed to evaluate the effect of mercury on the formation of biofilm by environmental (collected from urban stream water) and clinical isolates of Klebsiella pneumoniae. In addition, antibiotic resistance, virulence factors, and genetic diversity were investigated. Taxonomic identity of eight isolates (one reference, two clinical, and five environmental isolates) was performed by MALDI-TOF-MS, while the antibiotic susceptibility profile was assessed by the disc diffusion method. The ability to form biofilms was evaluated by culture on Congo red agar and by crystal violet staining. Biofilm structure was analyzed by scanning electron microscopy. The hydrophobicity profile and the presence of the virulence genes cps, fimH, and mrkD was investigated. The presence of merA and its relationship with antimicrobial resistance were also assessed. The identity of all isolates was confirmed by MALDI-TOF-MS, and different profiles of resistance to mercury and antibiotics as well as of biofilm formation were identified for the clinical and environmental isolates. All isolates were hydrophilic and positive for the virulence genes cps, fimH, and mrkD; only the clinical isolate K36-A2 was positive for merA. The diversity of the isolates was confirmed by ERIC-PCR, which revealed high heterogeneity among the isolates. In conclusion, the data demonstrate that the investigated isolates present different responses to exposure to Hg 2+ and correspond to distinct populations of K. pneumoniae disseminated in the investigated environment. The data obtained in this work will aid in understanding the mechanisms of survival of this pathogen under adverse conditions., (Copyright © 2018. Published by Elsevier Inc.)

In Brazil, DENV-1 introduced in the 80's, remained the prevalent serotype from 2012 to 2016. After its re-emergence in the country in 2009, the co-circulation of different viral lineages was identified, however, its transmission dynamics afterwards, was not fully characterized. In this study, we performed the continuous molecular surveillance after the reemergence period (2012 to 2016), covering the 30 years of circulation of DENV-1 in Brazil. Phylogenetic analysis allowed confirmation of the continued presence of genotype V, as well as three distinct co-circulating lineages. The molecular characterization of the E gene presented two new amino acid substitutions previously unidentified in the country. Phylogeographic analysis has shown that a large flow of migrations has occurred between Brazil and Argentina in the last 10 years.

Currently, Brazil lives a triple arboviruses epidemic (DENV, ZIKV and CHIKV) making the differential diagnosis difficult for health professionals. Here, we aimed to investigate chikungunya cases and the possible occurrence of co-infections during the epidemic in Amapá (AP) that started in 2014 when the first autochthonous cases were reported and in Rio de Janeiro (RJ) in 2016. We further performed molecular characterization and genotyping of representative strains. In AP, 51.4% of the suspected cases were confirmed for CHIKV, 71.0% (76/107). Of those, 24 co-infections by CHIKV/DENV, two by CHIKV/DENV-1, and two by CHIKV/DENV-4 were observed. In RJ, 76.9% of the suspected cases were confirmed for CHIKV and co-infections by CHIKV/DENV ( n = 8) and by CHIKV/ZIKV ( n = 17) were observed. Overall, fever, arthralgia, myalgia, prostration, edema, exanthema, conjunctival hyperemia, lower back pain, dizziness, nausea, retroorbital pain, and anorexia were the predominating chikungunya clinical symptoms described. All strains analyzed from AP belonged to the Asian genotype and no amino acid changes were observed. In RJ, the East-Central-South-African genotype (ECSA) circulation was demonstrated and no E1-A226V mutation was observed. Despite this, an E1-V156A substitution was characterized in two samples and for the first time, the E1-K211T mutation was reported in all samples analyzed.

In the 80s, dengue viruses type 1 and 4 (DENV-1 and 4) were isolated in North region of Brazil. However, it was only after the DENV-1 introduction in the state of Rio de Janeiro (RJ) in mid-1980s, that dengue became a nationwide public health problem. In 2009, this serotype re-emerged causing an explosive epidemic in the country. DENV-4 was first detected in RJ in 2011 and in 2012, and DENV-1 and 4 were co-circulating and responsible for a high number of cases notifications. Here, we describe the detection and molecular characterization of a DENV-1/4 co-infection in sample of 2012 in RJ.

Background: Dengue viruses (DENV) have emerged and reemerged in Brazil in the past 30 years causing explosive epidemics. The disease may range from clinically asymptomatic infections to severe and fatal outcomes. We aimed to describe the epidemiological, clinical and laboratorial aspects of the dengue fatal cases received by a Regional Reference Laboratory, Brazil in 30 years., Methods: A total of 1047 suspected fatal dengue cases were received from 1986 to 2015 and analyzed in the Laboratory of Flavivirus, FIOCRUZ. Suspected cases were submitted to viral detection, serological and molecular methods for cases confirmation. Influence of gender, age, serotype and type of infection (primary/secondary) on death outcome, as well the interactions between serotype and age or infection and age and type of infection were also studied., Results: A total of 359 cases (34.2%) were confirmed and DENV-1 (11.1%), DENV-2 (43.9%), DENV-3 (32.8%) and DENV-4 (13.7%) were detected. Overall, fatal cases occurred more often in primary infections (59.3%, p = 0.001). However, in 2008, fatal cases were mainly associated to secondary infections (p = 0.003). In 2008 and 2011, deaths were more frequent on children and those infected by DENV-2 presented a higher risk for fatal outcome. Moreover, children with secondary infections had a 4-fold higher risk for death., Conclusions: Dengue is a multifactorial disease and, factors such as viral strain/serotype, occurrence of secondary infections and co-morbidities may lead to a severe outcome. However, the high dengue incidence and transmission during epidemics, such as those observed in Brazil may overwhelm and collapse the public health services, potentially impacting on increased disease severity and mortality.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

Dengue is a worldwide problem characterized by a multifactorial pathogenesis. Considering the viral components, it is known that high viremia or high levels of the secreted nonstructural protein 1 (NS1) may be associated with a more severe disease. We aimed to characterize the NS1 antigenemia and viremia in dengue fatal and non-fatal cases, as potential markers of progression to a fatal outcome. NS1 antigenemia and viremia were determined in Brazilian dengue fatal cases ( n = 40) and non-fatal cases ( n = 40), representative of the four dengue virus (DENV) serotypes. Overall, the fatal cases presented higher NS1 levels and viremia. Moreover, the fatal cases from secondary infections showed significantly higher NS1 levels than the non-fatal ones. Here, irrespective of the disease outcome, DENV-1 cases presented higher NS1 levels than the other serotypes. However, DENV-2 and DENV-4 fatal cases had higher NS1 antigenemia than the non-fatal cases with the same serotype. The viremia in the fatal cases was higher than in the non-fatal ones, with DENV-3 and DENV-4 presenting higher viral loads. Viral components, such as NS1 and viral RNA, may be factors influencing the disease outcome. However, the host immune status, comorbidities, and access to adequate medical support cannot be ruled out as interfering in the disease outcome.

Chronic inflammatory process in the nasal mucosa is correlated with poor smell perception. Over-activation of immune cells in the olfactory epithelium (OE) is generally associated with loss of olfactory function, and topical steroidal anti-inflammatory drugs have been largely used for treating such condition. Whether this therapeutic strategy could directly affect the regenerative process in the OE remains unclear. In this study, we show that nasal topical application of dexamethasone (DEX; 200 or 800 ng/nostril), a potent synthetic anti-inflammatory steroid, attenuates OE lesion caused by Gram-negative bacteria lipopolysaccharide (LPS) intranasal infusion. In contrast, repeated DEX (400 ng/nostril) local application after lesion establishment limited the regeneration of olfactory sensory neurons after injury promoted by LPS or methimazole. Remarkably, DEX effects were observed when the drug was infused as 3 consecutive days regimen. The anti-inflammatory drug does not induce OE progenitor cell death, however, disturbance in mammalian target of rapamycin downstream signaling pathway and impairment of protein synthesis were observed during the course of DEX treatment. In addition, in vitro studies conducted with OE neurospheres in the absence of an inflammatory environment showed that glucocorticoid receptor engagement directly reduces OE progenitor cells proliferation. Our results suggest that DEX can interfere with the intrinsic regenerative cellular mechanisms of the OE, raising concerns on the use of topical anti-inflammatory steroids as a risk factor for progressive olfactory function impairment.

Background: The current triple epidemic caused by dengue, zika and chikungunya constitutes a serious health problem in Brazil. The aim of this study was to investigate acute samples (up to the 7 days of symptoms) from patients presenting acute fever syndrome suspected as arboviral infection and characterize the clinical and laboratorial profile during the co-circulation of dengue, zika and chikungunya in Campo Grande, Mato Grosso do Sul (MS), midwest region of Brazil., Methods: All suspected cases (n=134) were tested by using serological and molecular diagnostic tests including DENV, ZIKV and CHIKV RT-PCR, Dengue nonstructural protein 1 (NS1) antigen capture ELISA, anti- DENV IgM ELISA and anti-CHIKV IgM ELISA. In addition, clinical, hematological and biochemical parameters of infected patients were analyzed., Results:  It was observed that 79.1% of the blood samples were confirmed for ZIKV and/or DENV infection Of those, 38.0% patients were DENV monoinfected, 26.8% were ZIKV monoinfected and 13.4% were DENV/ZIKV co-infected. Seven patients presented Chikungunya IgM antibodies indicating a previous CHIKV infection. Common symptoms included fever, rash, arthralgia, myalgia, prostration, headache and conjunctivitis. Statistical analysis showed that pruritus and edema were associated with ZIKV infection while prostration and vomiting were more associated with dengue. Additionally, total protein and ALT levels were significantly different in DENV patients compared to ZIKV ones. Some DENV infected patients as well as co-infected needed hospitalization and venous hydration. Otherwise, most ZIKV infected patients presented mild clinical course. Among the pregnant women studied (n=11), three were ZIKV monoinfected while four were DENV monoinfected and two were DENV-1/ZIKV coinfected. In general, normal birth outcomes were observed except for the death due to respiratory insufficiency of one baby born to a mother coinfected with DENV-1/ZIKV., Conclusions:  Herein, we provide evidence of the co-circulation of DENV, ZIKV and CHIKV infections in the Campo Grande, MS, Brazil, with a high frequency of DENV-1/ZIKV coinfection. Laboratorial diagnosis poses a challenge where those arboviruses are endemic and differential diagnosis proved to imperative for cases characterization. The knowledge about disease severity during arbovirus coinfections is still scarce and there are several issues emphasizing the importance of adequate management of patients at risk areas.

In the large Zika virus (ZIKV) epidemic that occurred in Brazil in 2015, the intrauterine fetal exposure to ZIKV was associated with a significant risk of developing microcephaly and neurological disorders in the infected infants. ZIKV-associated disease has since been reported in 24 countries in the Americas. At present, definitive evidence is lacking regarding the intrauterine co-exposure to ZIKV and other viral infections and whether the coinfection impacts the risk of acquiring either infection or disease severity. Here, we provide evidence of intrauterine exposure to both ZIKV and human immunodeficiency virus (HIV) infections, causing congenital Zika syndrome in an HIV-exposed uninfected infant. Clinical, imaging and laboratory examinations of the pregnant woman and the newborn were performed. Histopathology, ZIKV/HIV-specific immunoassays, and ultrastructural evaluation of the placenta were performed. The Zika-asymptomatic, HIV-positive pregnant woman underwent ultrasounds revealing fetal cerebral ventriculomegaly, microcephaly, and brain atrophy. Her baby girl was born small for gestational age and with the neurological sequelae of congenital Zika syndrome. The evaluation of the abnormally large term placenta revealed severe damage to the maternal decidua and chorionic villi, cells positive for ZIKV-specific antigens but not for HIV antigens, and intracellular membranous clusters of virus-like particles approximately 25 nm in diameter. The rapid progression and severity of the congenital Zika syndrome may be related to the uncontrolled HIV disease in the mother. The poor inflammatory response observed in the placenta may have reduced the inherent risk of mother-to-child transmission of HIV.

Ae. aegypti is the main vector of dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. The transmission dynamics of these arboviruses, especially the arboviral circulation in the mosquito population during low and high transmission seasons in endemic areas are still poorly understood. We conducted an entomological survey to determine dengue infection rates in Ae. aegypti and Aedes albopictus. These collections were performed in 2012-2013 during a Rio de Janeiro epidemic, just before the introduction and spread of ZIKV and CHIKV in the city. MosquiTrap © and BG-Sentinel traps were installed in three fixed and seven itinerant neighborhoods each month over ten months. Mosquitoes were in supernatants pools tested and individually confirmed for DENV infection using RT-PCR. A total of 3053 Aedes mosquitos were captured and Ae. aegypti was much more frequent (92.9%) than Ae. albopictus (6.8%). Ae. aegypti females accounted for 71.8% of captured mosquitoes by MosquitTrap © and were the only species found naturally infected with DENV (infection rate=0.81%). Only one Ae. aegypti male, collected by BG-sentinel, was also tested positive for DENV. The peak of DENV-positive mosquitoes coincided the season of the highest incidence of human cases. The most common serotypes detected in mosquitoes were DENV-3 (24%) and DENV-1 (24%), followed by DENV-4 (20%), DENV-2 (8%) and DENV-1 plus DENV4 (4%), while 95% of laboratory-confirmed human infections in the period were due to DENV-4. These contrasting results suggest silent maintenance of DENV serotypes during the epidemics, reinforcing the importance of entomological and viral surveillance in endemic areas., (Copyright © 2017 Elsevier B.V. All rights reserved.)