Search

Purpose: Aedes aegypti mosquito-borne diseases have a significant impact on public health in Brazil. In this study, we investigated the presence of the Zika virus (ZIKV) and dengue virus (DENV) in serum and urine samples from symptomatic participants who attended an Emergency Care Unit located in a city in the northwestern region of São Paulo between February 2018 and April 2019., Methods: Serum and urine samples were collected from participants suspected of having arbovirus infection. After the extraction of viral RNA, viral detection was performed by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) (One-Step RT-qPCR)., Results: A total of 305 participants participated in this study. A total of 283 blood and 270 urine samples were collected. Of 305 patients, 36.4% (111/305) were positive for ZIKV, 43.3% (132/305) for DENV2, and 0.3% (1/305) for DENV1. Coinfection with ZIKV/DENV2 was observed in 13.1% of participants. If only serum samples were used, ZIKV detection would have decreased to 23.3% (71/305). Of all the participants included in the study, only one was suspected of having ZIKV infection based on clinical diagnosis, and the remaining participants were suspected of having DENV., Conclusion: By testing serum and urine samples, we increased the detection of both viruses and detected considerable levels of ZIKV and DENV-2 coinfection when compared to other studies. Additionally, we detected an unnoticed ZIKV outbreak in the city. These findings highlight the importance of the molecular diagnosis of arboviruses to aid public health surveillance and management strategies., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are important disease-causing agents worldwide. Currently, there are no antiviral drugs or vaccines approved to treat these viruses. However, peptides have shown great potential for new drug development. A recent study described (p-BthTX-I) 2 K [(KKYRYHLKPF) 2 K], a peptide derived from the Bothropstoxin-I toxin in the venom of the Bothrops jararacussu snake, showed antiviral activity against SARS-CoV-2. In this study, we assessed the activity of this peptide against CHIKV and ZIKV and its antiviral action in the different stages of the viral replication cycle in vitro. We observed that (p-BthTX-I) 2 K impaired CHIKV infection by interfering with the early steps of the viral replication cycle, reducing CHIKV entry into BHK-21 cells specifically by reducing both the attachment and internalization steps. (p-BthTX-I) 2 K also inhibited the ZIKV replicative cycle in Vero cells. The peptide protected the cells against ZIKV infection and decreased the levels of the viral RNA and the NS3 protein of this virus at viral post-entry steps. In conclusion, this study highlights the potential of the (p-BthTX-I) 2 K peptide to be a novel broad-spectrum antiviral candidate that targets different steps of the replication cycle of both CHIKV and ZIKV.

In the context of cancer predisposition syndromes, it is widely known that the correct interpretation of germline variants identified in multigene panel testing is essential for adequate genetic counseling and clinical decision making, in which variants of uncertain significance (VUS) are not considered actionable findings. Thus, their periodic re-evaluation using appropriate guidelines is notably important. In the present study, we compared the performance of the main variant classification guidelines (ACMG, Sherloc and ENIGMA) in variant reassessment, using as input a BRCA1/2 VUS case series (retrospective analysis) from Brazil, an ethnically diverse and admixed country with substantial challenges in VUS reclassification. As main findings, two of the 15 VUS analyzed were reclassified as likely pathogenic by the 3 guidelines, BRCA1 c.4987-3C > G (rs397509213) and BRCA2 c.7868A > G (rs80359012). Moreover, challenges in variant classification and reassessment are described and additional in silico data about structural impact of the variant BRCA2 c.7868A > G are provided. We hypothesize that the establishment of a framework to reassess VUS could improve this process in health centers that have not yet implemented this practice. Results of this study underscore that periodic monitoring of the functional, clinical, and bioinformatics data of a VUS by a multidisciplinary team are of utmost importance in clinical practice. When there is a specific guideline for a given gene, such as ENIGMA for BRCA1/2, it should be considered the first option for variant assessment. Finally, recruitment of VUS carriers and their relatives to participate in variant segregation studies and publication of VUS reclassification results in the international scientific literature should be encouraged., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Non-SARS-CoV-2 respiratory viral infections, such as influenza virus (FluV) and human respiratory syncytial virus (RSV), have contributed considerably to the burden of infectious diseases in the non-COVID-19 era. While the rates of co-infection in SARS-CoV-2-positive group (SCPG) patients have been determined, the burden of other respiratory viruses in the SARS-CoV-2-negative group (SCNG) remains unclear. Here, we conducted a cross-sectional study (São José do Rio Preto county, Brazil), and we collected our data using a meta-analysis to evaluate the pooled prevalence of FluV and RSV among SCNG patients. Out of the 901 patients suspected of COVID-19, our molecular results showed positivity of FluV and RSV in the SCNG was 2% (15/733) and 0.27% (2/733), respectively. Co-infection with SARS-CoV-2 and FluV, or RSV, was identified in 1.7% of the patients (3/168). Following our meta-analysis, 28 studies were selected ( n = 114,318 suspected COVID-19 patients), with a pooled prevalence of 4% (95% CI: 3-6) for FluV and 2% (95% CI: 1-3) for RSV among SCNG patients were observed. Interestingly, FluV positivity in the SCNG was four times higher (OR = 4, 95% CI: 3.6-5.4, p < 0.01) than in the SCPG. Similarly, RSV positivity was significantly associated with SCNG patients (OR = 2.9, 95% CI: 2-4, p < 0.01). For subgroup analysis, cold-like symptoms, including fever, cough, sore throat, headache, myalgia, diarrhea, and nausea/vomiting, were positively associated ( p < 0.05) with the SCPG. In conclusion, these results show that the pooled prevalence of FluV and RSV were significantly higher in the SCNG than in the SCPG during the early phase of the COVID-19 pandemic.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Background: Cellular entry of SARS-CoV-2 has been shown to rely on angiotensin-converting enzyme 2 (ACE2) receptors, whose expression in the testis is among the highest in the body. Additionally, the risk of mortality seems higher among male COVID-19 patients, and though much has been published since the first cases of COVID-19, there remain unanswered questions regarding SARS-CoV-2 impact on testes and potential consequences for reproductive health. We investigated testicular alterations in non-vaccinated deceased COVID-19-patients, the precise location of the virus, its replicative activity, and the immune, vascular, and molecular fluctuations involved in the pathogenesis., Results: We found that SARS-CoV-2 testicular tropism is higher than previously thought and that reliable viral detection in the testis requires sensitive nanosensors or RT-qPCR using a specific methodology. Through an in vitro experiment exposing VERO cells to testicular macerates, we observed viral content in all samples, and the subgenomic RNA's presence reinforced the replicative activity of SARS-CoV-2 in testes of the severe COVID-19 patients. The cellular structures and viral particles, observed by transmission electron microscopy, indicated that macrophages and spermatogonial cells are the main SARS-CoV-2 lodging sites, where new virions form inside the endoplasmic reticulum Golgi intermediate complex. Moreover, we showed infiltrative infected monocytes migrating into the testicular parenchyma. SARS-CoV-2 maintains its replicative and infective abilities long after the patient's infection. Further, we demonstrated high levels of angiotensin II and activated immune cells in the testes of deceased patients. The infected testes show thickening of the tunica propria, germ cell apoptosis, Sertoli cell barrier loss, evident hemorrhage, angiogenesis, Leydig cell inhibition, inflammation, and fibrosis., Conclusions: Our findings indicate that high angiotensin II levels and activation of mast cells and macrophages may be critical for testicular pathogenesis. Importantly, our findings suggest that patients who become critically ill may exhibit severe alterations and harbor the active virus in the testes., (© 2023. The Author(s).)

Ilheus fever is a mosquito-borne, poorly known tropical disease. We aimed to report the pooled rate of exposure to the Ilheus virus (ILHV) and clinical outcomes of infection to determine the epidemiological patterns of ILHV. We conducted a meta-analysis of 37 studies ( n = 17,722 individuals) from Latin America. The common clinical characteristics of ILHV infection were fever (82.3%), headache (52.9%), and myalgia (52.9%). Encephalitis complicated the course of the infection in 29.4% cases. Monotypic serological reactions detected a pooled rate of exposure of 2% to ILHV (95% CI: 1-2). Studies were mainly conducted in Brazil, with a pooled proportion of ILHV positivity of 8% (95% CI: 3-14). Males (12%) had higher rates of seropositivity than females (7%) and had high chances of ILHV infection (OR: 1.7, 95% CI: 1.2-2.5). Seropositivity increased with age, from 2% (95% CI: 2-3) among people aged 0-14 years to 8% (95% CI: 6-10) among people aged 15-64 years. Our analysis indicated a low and relatively constant burden of ILHV in Latin America. More research is needed to evaluate and innovate serological assays for ILHV to better estimate the burden and dynamics of epidemiological changes in ILHV infection in different regions.

The chronic hepatitis C (CHC) treatment is currently based on the use of direct-acting antivirals (DAAs), and patients infected with hepatitis C virus genotype 3 (GT3) have emerged as a more difficult-to-cure population. The NS5A inhibitor daclatasvir (DCV) and sofosbuvir (SOF), an NS5B viral polymerase inhibitor, are among the drugs that compose more effective and safer treatment regimens. The virus genetic variability is related to resistance-associated substitutions (RASs) that adversely impact DAAs effectiveness. The aims of this study were to analyze the association of NS5A and NS5B RASs and other clinical factors with DAAs regimens effectiveness in patients with GT3 CHC infection. This was a prospective cohort study performed in a Brazilian university hospital. Individuals older than 18 years with GT3 CHC treated with SOF + DCV ± ribavirin (RBV) or SOF + peginterferon (PEG) + RBV were included. Blood samples were collected at baseline and post-treatment. A total of 121 patients were included. Sustained virological response rates were 87.6% for the SOF + DCV ± RBV group and 80.0% for the SOF + PEG + RBV arm. Cirrhosis, prior treatment with interferon/PEG + RBV, and baseline NS5A RAS were associated with higher risk of treatment failure. The NS5A analysis suggested that A30K, Y93H, and RAS at site 62 were related to failure. Interestingly, a likely compensatory effect was shown between A30K and A62T. Emergence of Y93H was always associated with RAS at position 62. The RASs dynamics comprehension is an important tool to indicate more effective treatment for GT3 patients., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2022 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Poor vigor at birth has been associated with reduced IgG absorption from colostrum and a reduced vitality in neonatal dairy calves. Some natural compounds, such as green tea extract, may improve vitality in compromised calves. The objective of this randomized controlled trial was to evaluate the potential of supplementing a green tea extract (15 mL) to calves to improve vigor and activity behavior for the first 72 h postnatal. Also, this study aimed to investigate the influence of green tea extract supplementation on calf serum IgG concentration and the apparent efficiency of absorption (AEA) of colostral IgG. Holstein calves (n = 24) weighing 42.49 ± 1.07 kg postnatal received a complete random assignment at 3 h of one 15-mL dose of green tea extract (Calf Perk, TechMix) or distilled water orally before tube feeding colostrum replacer (Premolac Plus IgG, Zinpro) at 4 h postnatal. Two observers assessed for calving time and dystocia by live video stream to retrieve all calves within 2 h postnatal. One veterinarian performed a baseline vigor assessment based on heart rate and response to stimuli on all calves at 2.5 h, before colostrum feeding at 3.75 h, as well as at 24, 48, and 72 h postnatal. Calf blood samples were taken to assess total IgG by radial immunodiffusion assay at 2.5, 6, 12, 24, 48, and 72 h. Calf vitality was also observed continuously by video for all calves to determine whether treatment was associated with attempts to stand, lying time, and exploration of their pen environment for the first 24 h. We used an ordinal logistic model to evaluate the odds of green tea extract improving a calf's vigor category from 2.5 h postnatal to 72 h of age. Vigor score was categorized as abnormal (≤4), average (5), or alert (≥6), with hour as a fixed effect. We also ran mixed linear models to evaluate the effect of extract on total IgG and AEA, with time and dystocia as fixed effects. Five dystocia calves were enrolled (2 control, 3 extract), but assistance was minor (e.g., manual assistance and all were assisted within 1 h). Baseline vigor scores and baseline total IgG were not different between groups. Vigor score category was not associated with green tea extract supplementation (odds ratio 1.17; 95% CI: 0.43-3.15) but increased with time compared with controls. We observed no association of treatment with total IgG or AEA in the calves, suggesting green tea extract does not compromise IgG absorption. Calf vitality, lying behavior, and exploratory behavior were not associated with green tea extract treatment. Our findings suggest that green tea extract supplementation does not affect AEA and serum IgG concentration in calves. Future research should evaluate whether green tea extract improves vitality in calves experiencing severe dystocia., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Wastewater-based epidemiology (WBE) is a tool involving the analysis of wastewater for chemicals and pathogens at the community level. WBE has been shown to be an effective surveillance system for SARS-CoV-2, providing an early-warning-detection system for disease prevalence in the community via the detection of genetic materials in the wastewater. In numerous nation-states, studies have indicated the presence of SARS-CoV-2 in wastewater. Herein, we report the primary time-course monitoring of SARS-CoV-2 RNA in wastewater samples in São José do Rio Preto-SP/Brazil in order to explain the dynamics of the presence of SARS-CoV-2 RNA during one year of the SARS-CoV-2 pandemic and analyze possible relationships with other environmental parameters. We performed RNA quantification of SARS-CoV-2 by RT-qPCR using N1 and N2 targets. The proportion of positive samples for every target resulted in 100% and 96.6% for N1 and N2, respectively. A mean lag of -5 days is observed between the wastewater signal and the new SARS-CoV-2-positive cases reported. A correlation was found between the air and wastewater temperatures and therefore between the SARS-CoV-2 viral titers for N1 and N2 targets. We also observed a correlation between SARS-CoV-2 viral titers and media wastewater flow for the N1 target. In addition, we observed higher viral genome copies within the wastewater samples collected on non-rainy days for the N1 target. Thus, we propose that, based on our results, monitoring raw wastewater may be a broadly applicable strategy that might contribute to resolving the pressing problem of insufficient diagnostic testing; it may represent an inexpensive and early-warning method for future COVID-19 outbreaks, mainly in lower- and middle-income countries.

Studies have shown that β-glucans extracted from the cell wall of cereals, algae, and yeasts have been associated with improved immune function. However, it is unknown whether algae β-glucan supplementation affects the performance, blood metabolites, or cell counts of immune cells in dairy calves. The objective of this randomized clinical trial was to evaluate whether supplementation of β-glucans to milk replacer in dairy calves fed 6 L/d improved growth performance and fecal status and altered the blood metabolite profile. In this trial, we enrolled Holstein calves (n = 34) at birth (body weight 36.38 ± 1.33 kg; mean ± standard deviation) to receive, from 1 d of age, either 2 g/d algae β-glucans mixed into 6 L/d of milk replacer (22.4% crude protein and 16.2% fat) or an unsupplemented milk replacer (control). The calves were blocked in pairs according to birth weight, sex, and date of birth (up to 5 d difference). Calves were housed individually, and calf starter (24.7% crude protein and 13.9% neutral detergent fiber) was offered ad libitum based on orts of the previous day until 56 d of age (end of the trial). Body weight was measured weekly, and health checks and daily fecal consistency were evaluated daily in every calf by the same observer. Calves with 2 consecutive days of loose feces that sifted through bedding were considered diarrhea positive. We used a linear mixed effects model to evaluate the effects of β-glucan supplementation fed during the preweaning period on performance (average daily gain), final weight, feed efficiency (FE), white blood cell count, and selected blood metabolites, repeated by time. A generalized linear mixed effects model was also run to evaluate the likelihood of a diarrhea bout in the first 28 d of life, controlling for the calf as the subject with a logistic distribution. We included age, serum total protein at 48 h, and birth weight as covariates. At 56 d, β-glucan-supplemented calves weighed more than control calves (56.3 vs. 51.5 kg). Treatment had no effect on total starter intake, but there was a treatment by age interaction for FE, with greater FE for β-glucan-supplemented calves in wk 3 and 5 of age. There was only a tendency for average daily gain to be greater in supplemented calves than in control calves for the duration of the study. Furthermore, control calves had 14.66 [95% confidence interval (95% CI): 9.87-21.77] times greater odds of having a diarrheal bout than β-glucan-supplemented calves. Control calves had 12.70 (95% CI: 8.82-18.28) times greater odds of having an additional day with an abnormal fecal score compared with β-glucan-supplemented calves, suggesting that supplementation ameliorated diarrhea severity. We found no association of treatment with concentrations of serum total protein, albumin, creatinine, or glucose during the preweaning period. Our findings suggest that dietary supplementation of 2 g/d of algae β-glucans to milk replacer improved fecal status and may affect growth, as evidenced by a higher weaning weight, compared with control calves. Future studies should explore the effect of algae β-glucans on lower-gut physiology and digestibility in dairy calves., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Sexual transmission of Zika virus (ZIKV), an important arbovirus, and the virus persistence in semen raise several questions about how and where it circulates in the male reproductive system (MRS). Several studies reported detection of the virus in testes, epididymis, and prostate at 5 days post-infection (dpi) or more in animal models. In the present study, we investigated the interactions of ZIKV with mouse MRS using the AG129 strain, a ZIKV permissive immunodeficient mouse strain, at two dpi. Viral RNA was detected in blood, testes, epididymis, and prostatic complexes (prostate and seminal vesicles). Immunohistochemical (IHC) analyses, based on the envelope protein, showed an early infection in organs of MRS since ZIKV positive antigens were detected in cells within or surrounding blood vessels, Sertoli, and germ cells in testes and epithelial cells in epididymis and prostate. Positive antigens for NS5 protein, the virus RNA-dependent RNA polymerase, were also detected by IHC in these organs and circulating leukocytes, suggesting that the virus replicates in these sites as early as 2 days post-infection. Analysis of the early stages of ZIKV infection in MRS may improve the current knowledge about this issue and contribute to the development of therapies directed to the infection at this site., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: The emergence of the Brazilian variant of concern, Gamma lineage (P.1), impacted the epidemiological profile of COVID-19 cases due to its higher transmissibility rate and immune evasion ability., Methods: We sequenced 305 SARS-CoV-2 whole-genomes and performed phylogenetic analyses to identify introduction events and the circulating lineages. Additionally, we use epidemiological data of COVID-19 cases, severe cases, and deaths to measure the impact of vaccination coverage and mortality risk., Results: Here we show that Gamma introduction in São José do Rio Preto, São Paulo, Brazil, was followed by the displacement of seven circulating SARS-CoV-2 variants and a rapid increase in prevalence two months after its first detection in January 2021. Moreover, Gamma variant is associated with increased mortality risk and severity of COVID-19 cases in younger age groups, which corresponds to the unvaccinated population at the time., Conclusions: Our findings highlight the beneficial effects of vaccination indicated by a pronounced reduction of severe cases and deaths in immunized individuals, reinforcing the need for rapid and massive vaccination., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2022.)

The present study aimed to estimate covariance components of milk fatty acids (FA) and to compare the genomic estimated breeding values under general and heat-stress effects. Data consisted of 38,762 test-day records from 6,344 Holstein cows obtained from May 2012 through January 2018 on 4 dairy herds from Brazil. Single-trait repeatability test-day models with random regressions as a function of temperature-humidity index values were used for genetic analyses. The models included contemporary groups, parity order (1-6), and days in milk classes as fixed effects, and general and thermotolerance additive genetic and permanent environmental as random effects. Notably, differences in heritability estimates between environments (general and heat stress) increased (0.03 to 0.06) for unsaturated FA traits, such as unsaturated, monounsaturated, and polyunsaturated, at higher heat-stress levels. In contrast, heritability estimated between environments for saturated FA traits, including saturated FA, palmitic acid (C16:0), and stearic acid (C18:0) did not observe significant differences between environments. In addition, our study revealed negative genetic correlations between general and heat-stress additive genetic effects (antagonistic effect) for the saturated FA, C16:0, C18:0, and C18:1, which ranged from -0.007 to -0.32. Spearman's ranking correlation between genomic estimated breeding values ranged from -0.27 to 0.99. Results indicated a moderate to strong interaction of genotype by the environment for most FA traits comparing a heat-stress environment with thermoneutral conditions. Our findings point out novel opportunities to explore the use of FA milk profile and heat-stress models., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Purpose: Studies show that around 80% of Zika virus (ZIKV) infections are asymptomatic. The present study tested urine samples from volunteers, unsuspected of arboviral infection, which attended an emergency care unit (ECU) in Mirassol, Brazil, from March 2018 to April 2019., Methods: The volunteers were divided into two groups. The first group was composed of outpatients who were not suspected to have an arbovirus infection. This first group was subdivided into two subgroups: outpatients with and without arbovirus-like symptoms. The second group consisted of companions of outpatients treated at the ECU. The second group was also subdivided into two subgroups: totally asymptomatic individuals and those who had arbovirus-like symptoms. RNA was extracted from urine samples, followed by RT-qPCR for ZIKV., Results: We found that 11% (79/697) of the samples tested positive for ZIKV-RNA. Among the ZIKV-RNA-positive individuals, 16.5% (13/79) were companions, of which 61.5% (8/13) were totally asymptomatic and 38.5% (5/13) reported symptoms that could be suggestive of arbovirus infection. In addition, 83.5% (66/79) of the ZIKV-RNA-positive individuals were outpatients without a clinical diagnosis of arbovirus. Of these undiagnosed ZIKV-RNA-positive outpatients, 47% (31/66) had no arbovirus-related symptoms., Conclusion: Our study shows the effectiveness of urine as a non-invasive sample to detect the incidence of ZIKV infection. We also highlight the importance of ZIKV molecular diagnosis to aid public health surveillance and prevention of congenital Zika syndrome and other ZIKV-associated diseases., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Knowledge about how the COVID-19 pandemic can affect aquatic wildlife is still extremely limited, and no effect of SARS-CoV-2 or its structural constituents on invertebrate models has been reported so far. Thus, we investigated the presence of the 2019-new coronavirus in different urban wastewater samples and, later, evaluated the behavioral and biochemical effects of the exposure of Culex quinquefasciatus larvae to two SARS-CoV-2 spike protein peptides (PSPD-2002 and PSPD-2003) synthesized in our laboratory. Initially, our results show the contamination of wastewater by the new coronavirus, via RT-qPCR on the viral N1 gene. On the other hand, our study shows that short-term exposure (48 h) to a low concentration (40 μg/L) of the synthesized peptides induced changes in the locomotor and the olfactory-driven behavior of the C. quinquefascitus larvae, which were associated with increased production of ROS and AChE activity (cholinesterase effect). To our knowledge, this is the first study that reports the indirect effects of the COVID-19 pandemic on the larval phase of a freshwater invertebrate species. The results raise concerns at the ecological level where the observed biological effects may lead to drastic consequences., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Patients with Coronavirus Disease 2019 (COVID-19) may present high risk features during hospitalization, including cardiovascular manifestations. However, less is known about the factors that may further increase the risk of death in these patients., Methods: We included patients with COVID-19 and high risk features according to clinical and/or laboratory criteria at 21 sites in Brazil from June 10th to October 23rd of 2020. All variables were collected until hospital discharge or in-hospital death., Results: A total of 2546 participants were included (mean age 65 years; 60.3% male). Overall, 70.8% were admitted to intensive care units and 54.2% had elevated troponin levels. In-hospital mortality was 41.7%. An interaction among sex, age and mortality was found (p = 0.007). Younger women presented higher rates of death than men (30.0% vs 22.9%), while older men presented higher rates of death than women (57.6% vs 49.2%). The strongest factors associated with in-hospital mortality were need for mechanical ventilation (odds ratio [OR] 8.2, 95% confidence interval [CI] 5.4-12.7), elevated C-reactive protein (OR 2.3, 95% CI 1.7-2.9), cancer (OR 1.8, 95 %CI 1.2-2.9), and elevated troponin levels (OR 1.8, 95% CI 1.4-2.3). A risk score was developed for risk assessment of in-hospital mortality., Conclusions: This cohort showed that patients with COVID-19 and high risk features have an elevated rate of in-hospital mortality with differences according to age and sex. These results highlight unique aspects of this population and might help identifying patients who may benefit from more careful initial surveillance and potential subsequent interventional therapies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)

The emergence of several SARS-CoV-2 lineages presenting adaptive mutations is a matter of concern worldwide due to their potential ability to increase transmission and/or evade the immune response. While performing epidemiological and genomic surveillance of SARS-CoV-2 in samples from Porto Ferreira-São Paulo-Brazil, we identified sequences classified by pangolin as B.1.1.28 harboring Spike L452R mutation, in the RBD region. Phylogenetic analysis revealed that these sequences grouped into a monophyletic branch, with others from Brazil, mainly from the state of São Paulo. The sequences had a set of 15 clade defining amino acid mutations, of which six were in the Spike protein. A new lineage was proposed to Pango and it was accepted and designated P.4. In samples from the city of Porto Ferreira, P.4 lineage has been increasing in frequency since it was first detected in March 2021, corresponding to 34.7% of the samples sequenced in June, the second in prevalence after P.1. Also, it is circulating in 30 cities from the state of São Paulo, and it was also detected in one sample from the state of Sergipe and two from the state of Rio de Janeiro. Further studies are needed to understand whether P.4 should be considered a new threat., 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 © 2021 Bittar, Possebon, Ullmann, Geraldini, da Costa, de Almeida, da S. Sanches, Nascimento-Júnior, Cilli, Artico Banho, Campos, Ferreira, Sacchetto, da Silva, Parra, Moraes, da Costa, Vasconcelos, Spilki, Nogueira, Rahal and Araujo Jr.)

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome caused by pathogenic germline variants in the TP53 gene, characterized by a predisposition to the development of a broad spectrum of tumors at an early age. The core tumors related to LFS are bone and soft tissue sarcomas, premenopausal breast cancer, brain tumors, adrenocortical carcinomas (ACC), and leukemias. The revised Chompret criteria has been widely used to establish clinical suspicion and support TP53 germline variant testing and LFS diagnosis. Information on TP53 germline pathogenic variant (PV) prevalence when using Chompret criteria in South America and especially in Brazil is scarce. Therefore, the aim of this study was to characterize patients that fulfilled these specific criteria in southern Brazil, a region known for its high population frequency of a founder TP53 variant c.1010G>A (p.Arg337His), as known as R337H. TP53 germline testing of 191 cancer-affected and independent probands with LFS phenotype identified a heterozygous pathogenic/likely pathogenic variant in 26 (13.6%) probands, both in the DNA binding domain (group A) and in the oligomerization domain (group B) of the gene. Of the 26 carriers, 18 (69.23%) were R337H heterozygotes. Median age at diagnosis of the first tumor in groups A and B differed significantly in this cohort: 22 and 2 years, respectively (P = 0.009). The present study shows the clinical heterogeneity of LFS, highlights particularities of the R337H variant and underscores the need for larger collaborative studies to better define LFS prevalence, clinical spectrum and penetrance of different germline TP53 pathogenic variants., Competing Interests: The authors have declared that no competing interests exist.

New variants of SARS-CoV-2 Alpha (B.1.1.7); Beta (B.1.351) Gamma (P.1) and Delta (B.1.617.2) quickly spread in the UK, South Africa, Brazil and India, respectively. To address whether mutations in SARS-CoV-2 RBD spike protein could affect virus infectivity, peptides containing RBD amino acids mutations have been constructed and interacted with human ACE2 by computational methods. Our results suggest that mutations in RBD amino acids K417, E484, L452, T478 and N501 are expressively increasing the affinity of this protein with human angiotensin-converting enzyme 2 (ACE2), consequently, variants Alpha (B.1.1.7), Beta (B1.351), Gamma (P.1) and Delta (B.1.617.2) could be more infective in human cells compared with SARS-CoV-2 isolated in Wuhan-2019 and the Gamma and Delta variants could be the most infective among them., Competing Interests: The authors declare that there is no interest conflict., (© 2021 The Author(s).)

Infection with distinct Zika virus (ZIKV) strains in in vitro and in vivo models has demonstrated that the host's response to infection is strain-dependent. There has been no analysis of the impact of infection with different ZIKV strains on miRNA expression in human cells. We investigated miRNA expression in PNT1A cells upon infection with an African ZIKV strain (MR766) and a Brazilian ZIKV strain (ZIKV BR ) using PCR array. Sixteen miRNAs were modulated in PNT1A cells: six miRNAs were modulated by both strains, while a set of ten miRNAs were modulated exclusively by ZIKV BR infection. In silico analysis showed that nine significant KEGG pathways and eight significant GO terms were predicted to be enriched upon ZIKV BR infection, and these pathways were related to cancer, environmental information processing, metabolism, and extracellular matrix. Differential modulation of miRNA expression suggests that distinct strains of ZIKV can differentially modulate the host response through the action of miRNAs.

Lysolecithin is an antiinflammatory emulsifier associated with improved apparent digestibility of total dietary fat and improved feed efficiency in dairy cattle. However, it is unknown if lysolecithin (LYSO) improves performance in calves. Moreover, since many conventional milk replacers use vegetable-sourced fat (e.g., palm oil), nutrient absorption and fecal score may be affected in neonatal calves. Thus, the objective of this study was to evaluate the effects of LYSO supplemented in milk replacer on performance, metabolites, and gut health of preweaned dairy calves. Holstein calves (n = 32) with adequate passive transfer were assigned in pairs (16 blocks) balanced by birth weight, date of birth, and sex at 1 d of age to randomly receive either LYSO (mixed in 2 milk replacer feedings at a rate of 4 g/d Lysoforte, Kemin Industries Inc., Des Moines, IA) or a milk replacer control (nothing added). Both treatments were fed 6 L/d milk replacer [22.5% crude protein, 16.2% crude fat (vegetable oil fat source) on a dry matter basis with 14% solids] by bucket in 2 daily feedings for 56 d. Calves were individually housed in wooden hutches and offered a commercial calf starter (24.6% crude protein and 13.9% neutral detergent fiber) and water by bucket ad libitum. Feed refusals and calf health was assessed daily. Weights and blood metabolites (glucose, total serum protein, albumin, creatinine, triglycerides, and cholesterol) were sampled weekly, and calves completed the study before weaning at 56 d of age. The effects of LYSO on calf average daily gain, feed efficiency, and blood metabolites were evaluated using a linear mixed model with time as a repeated measure, calf as the subject, and block as a random effect in SAS (SAS Institute Inc., Cary, NC). The effect of LYSO to improve the odds of abnormal fecal score was evaluated using a logistic model. Supplementation of LYSO increased average daily gain (control 0.28 ± 0.03 kg; LYSO 0.37 ± 0.03 kg; least squares means ± standard error of the mean) and increased feed efficiency (gain-to-feed; control 0.25 ± 0.03; LYSO 0.32 ± 0.03). Similarly, LYSO calves had a higher final body weight at d 56 (control 52.11 ± 2.33 kg; LYSO 56.73 ± 2.33 kg). Interestingly, total dry matter intake was not associated with LYSO despite improved average daily gain (total dry matter intake control 1,088.7 ± 27.62 g; total dry matter intake LYSO 1,124.8 ± 27.62 g). Blood glucose, albumin, creatinine, triglycerides, and cholesterol were not associated with LYSO. Indeed, only total serum protein had a significant interaction with LYSO and age at wk 5 and 6. Moreover, control calves had a 13.57 (95% confidence interval: 9.25-19.90) times greater odds of having an abnormal fecal score on any given day during the diarrhea risk period from d 1 to 28. The inclusion of LYSO as an additive in milk replacer in a dose of 4 g/d may improve performance, and calf fecal score, preweaning. Further research should investigate the mechanisms behind the effects of LYSO on fat digestibility in calves fed 6 L/d of milk replacer with vegetable-sourced fat., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs.

Tropical grass hay feeding is related to improved ruminal health; however, it may decrease energy intake. On the other hand, whole-flint corn grain may be an alternative fiber source in the diet of dairy calves. Forty-two Holstein calves were used in a randomized block design, considering sex, birth date, and weight at 21 d of age, when the supply of whole-flint corn grain or tropical grass hay started. Three component-fed solid diets were compared: (1) starter concentrate only during the preweaning and starter concentrate with free choice of chopped Tifton-85 hay postweaning (SC), (2) starter concentrate with free choice of chopped Tifton-85 hay pre- and postweaning (SCH), and (3) starter concentrate with free choice of whole-flint corn grain pre- and postweaning (SCW). The animals were evaluated from 21 to 84 d of age. Calves were managed equally during the first 21 d, fed with 6 L/d of whole milk and a commercial starter concentrate (46% nonfiber carbohydrates, small particles, and pelleted) ad libitum. After that, milk feeding was reduced to 4 L/d until gradual weaning at 56 d of age. At 56 d of age, 4 animals per treatment were randomly chosen to be slaughtered for digestive tract weight evaluation and to collect tissue for histological analysis of the ruminal wall, duodenum, and cecum, whereas the other 30 animals were weaned and evaluated for a further 22 d when the SC diet also received hay ad libitum. Feed intake was measured daily. Weight gain and metabolic indicators of intermediate metabolism were evaluated weekly. Ruminal fluid was collected at wk 6, 8, 10, and 12 of age. The SCH diet increased the total and starter dry matter intake, and consequently, the average daily gain and body weight at 56 d of age. The SCW diet promoted an increase in propionate and decreased acetate-to-propionate ratio. Morphometric variables were affected by the SCH diet. The postweaning performance was unaffected by solid diets; however, the SCW diet decreased ruminal and fecal pH. Feeding hay, starting at 21 d of age, can stimulate early solid diet intake, promoting better performance and ruminal and intestinal development, when a highly fermentable and small particle pelleted starter is fed., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: The World Health Organization estimates that 1% of the world population (71 million) is infected with hepatitis C virus (HCV). In 2015, three direct-acting antivirals (DAAs), simeprevir (SMV), sofosbuvir (SOF) and daclatasvir (DCV) were included in the Brazilian protocol for the treatment of chronic hepatitis C. Despite the fact that the use of these drugs is associated with higher treatment response rates and with lower incidence of side effects, studies have shown the association between the presence of viral resistance mutations and the failure of pharmacological treatment., Aim: This way, this study aimed to evaluate the safety and effectiveness of treatment for HCV genotypes 1a and 1b infected patients with these DAAs, also analyzing the occurrence and prevalence of baseline resistance associated substitutions (RAS), observing the impact of these mutations into the treatment success., Methods: Clinical data were collected from all the 262 HCV infected patients included for comparative analysis, while serum samples collected from 144 of these individuals, before treatment, were submitted to molecular biology approaches for mutation analysis into NS3, NS5A and NS5B regions., Results: Regarding the treatment regimens, 49.6% of the patients received SOF+DCV±ribavirin and 50.4% used SOF+SMV±ribavirin. The sustained virological response at 12 weeks post-treatment (SVR12) rate was 92.7% (93.9% for SOF plus DCV and 91.7% for SOF plus SMV). No clinical or laboratorial factor was statistically associated with SVR. The most common adverse reactions were haematological events, nausea/vomiting, headache and asthenia. Out of 144 blood samples, 70 (48.6%) had detected RAS, 34.8% treated with SOF+DCV±ribavirin and 61.3% SOF+SMV±ribavirin. The resistance mutations against SMV were detected into NS3: substitutions G122S (28%), I170V (22.7%), Y56F (17.3%) and V132I (14.7%). The mutations against DCV R30Q (9.1%), P58H (6.1%) and Q62E (6.1%) were observed into NS5A, and for SOF the mutations A421V (10.6%), L159F (6.4%) and C316N (6.4%) were present inside NS5B viral protein. Four patients did not reach SVR, three of them presented viruses carrying RAS (1 treated with SOF+DCV and 2 with SOF+SMV). Some of these mutations, like R30Q (present in relapsing samples) and L159F, are well known by their influence on antiviral resistance, while others, like C316N, have a compensatory effect on viral fitness, maintaining these baseline RAS., Conclusion: The use of treatment regimens composed of SOF and DCV or SOF and SMV showed a high SVR rate, despite of a high rate of RAS, and a good tolerability profile in patients with HCV genotype 1. However, the high occurrence of baseline RAS observed in this casuistic is still a concern and studies like this show the necessity to understand how they are maintained in the population and to direct more efficiently the use of DAAs., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The weaning process may cause intense stress for dairy calves, even when low volumes of liquid diet are fed. Management tools that increase the intake of solid feeds, such as gradual weaning, can provide better physiological and metabolic conditions through better ruminal development, leading to better adaptation to ruminant metabolism and aiding in stress mitigation. The objective of this study was to evaluate the effects of 2 weaning protocols and 2 levels of concentrate intake on the performance and physiological and behavioral variables related to stress in dairy calves. Thirty-six newborn male Holstein calves were used in a randomized block design with a 2 × 2 factorial arrangement: 2 weaning strategies, abrupt or gradual, and 2 levels of concentrate intake at 5 wk of age, high (>350 g/d) or low (≤350 g/d). Calves were equally managed until they were 5 wk of age and then grouped according to concentrate intake. Statistical analyzes were performed using the MIXED procedure of SAS software (SAS Institute Inc., Cary, NC), and no significant interaction was observed between studied factors (weaning method and starter intake level); therefore, we considered each factor separately and their interactions with age. The highest dry matter intake and concentration of β-hydroxybutyrate were recorded for animals with a high level of starter intake independent of the weaning method. Structural growth (cm/wk) and average daily gain were superior for calves with high starter intake, but weaning method had no effect. The gradual weaning protocol increased the time eating starter, regardless of the level of concentrate intake. Even animals with low concentrate intake that were weaned abruptly showed levels of cortisol and acid-soluble glycoprotein within normal physiological levels. Apparently, other factors besides the milk supply affect the starter intake level of calves in a conventional feeding program. The adoption of gradual weaning is not effective in improving performance when a calf has low intake 3 wk before weaning is complete, but it reduced vocalization on d 2 postweaning., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Bats are flying mammals distributed worldwide known to host several types of Coronavirus (CoV). Since they were reported as the probable source of spillover of highly pathogenic CoV into the human population, investigating the circulation of this virus in bats around the world became of great importance. We analyzed samples from 103 bats from two distinct regions in Brazil. Coronavirus from the Alphacoronavirus genus was detected in 12 animals, 11 from São José do Rio Preto-SP region and 1 from Barreiras-BA region, resulting in a prevalence of 17.18% and 2.56% respectively. The virus was detected not only in intestines but also in lungs and liver. Phylogenetic analysis based on nsP12 genomic region suggests that the sequences group according to host family and sampling location. Studies on the circulation of these viruses in bats remain important to understand the ecology and evolutionary relationship of these pathogens.

Zika virus (ZIKV) is a mosquito-transmitted Flavivirus, originally identified in Uganda in 1947 and recently associated with a large outbreak in South America. Despite extensive efforts there are currently no approved antiviral compounds for treatment of ZIKV infection. Here we describe the antiviral activity of diarylamines derived from anthranilic acid (FAMs) against ZIKV. A synthetic FAM (E3) demonstrated anti-ZIKV potential by reducing viral replication up to 86%. We analyzed the possible mechanisms of action of FAM E3 by evaluating the intercalation of this compound into the viral dsRNA and its interaction with the RNA polymerase of bacteriophage SP6. However, FAM E3 did not act by these mechanisms. In silico results predicted that FAM E3 might bind to the ZIKV NS3 helicase suggesting that this protein could be one possible target of this compound. To test this, the thermal stability and the ATPase activity of the ZIKV NS3 helicase domain (NS3 Hel ) were investigated in vitro and we demonstrated that FAM E3 could indeed bind to and stabilize NS3 Hel .

Propolis is a natural product produced by bees and sold commercially. It is well known for its antioxidant and anti-inflammatory effects and the ability to improve immune system functions in humans and animals. Many of its positive effects can contribute positively to animal productivity. The aim of this study was to evaluate possible beneficial effects of red propolis supplementation on growth performance, metabolism, and health status of dairy calves during the preweaning phase. Thirty-two newborn calves were individually housed, with free access to water and concentrate, and fed 6 L/d of whole milk. Animals were distributed in 2 treatments: (1) control and (2) supplementation with 4 mL/d of red propolis ethanolic extract (30%; EEP) in whole milk. Feed consumption and fecal and health scores were monitored daily from 1 d of age to 56 d. Calves were weighed and measured weekly. Blood samples were collected weekly, 2 h after morning milk feeding, for determination of glucose, total serum protein, urea, lactate, β-hydroxybutyrate, total iron binding capacity, and total leukocytes (white blood cells) and erythrocytes (red blood cells). Starter feed intake, daily weight gain, and body measurements were not affected by the EEP supplementation. Blood parameters were also not affected, with the exception for the red blood cell counts, which were lower in animals supplemented with EEP. Supplementation with EEP significantly decreased fecal score, days with diarrhea, and veterinary treatments of diarrhea. These results indicate that red propolis supplementation has potential to improve calf health and reduce the incidence of diarrhea and, as a consequence, the use of antibiotics in calf rearing systems., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Condyloma acuminata (CA), or genital warts, are benign proliferative epidermal or mucous lesions that are caused by infection with human papillomavirus (HPV), mainly the low-risk types 6 and 11. HPV variants are defined as viral sequences that share identity in the nucleotide sequence of the L1 gene greater than 98%. Based on this criterion, HPV6 and 11 variant lineages have been studied, and there are ongoing attempts to correlate these genetic variants with different clinical findings of infection. Therefore, the aims of this study were to detect variants and nucleotide alterations present in the E6 regions of HPV types 6 and 11 found in CA samples, to correlate the HPV presence with the clinical-pathological data of the patients, and to determine phylogenetic relationships with variants from other places in the world. The E6 regions of 25 HPV6 samples and 7 HPV11 samples from CA were amplified using PCR with specific primers. The products were ligated to a cloning vector and five colonies of each sample were sequenced to observe the nucleotide alterations. Twelve samples were identified as the HPV6B3 variant, presenting the mutation (guanine) G474A (adenine), and one of them also showed the mutation (thymine) T369G. The other 13 patients were positive for HPV6B1 without nucleotide alterations. In the analysis of the HPV11 samples, all patients showed the mutations T137C and (cytosine) C380T. One patient also presented the nucleotide alteration T410C. None of the mutations found in the 32 analyzed samples resulted in amino acid changes. Patient age, local occurrence, and HIV infection did not show significant association with HPV infection. Besides, the data found in this study did not show a relationship with the geographical region of isolation when compared to other data from different regions of the world. In this way, despite the nucleotide alterations found, it was not possible to observe amino acid changes and variants grouping according to geographical region.

Zika virus (ZIKV) has been associated with serious health conditions, and an intense search to discover different ways to prevent and treat ZIKV infection is underway. Berberine and emodin possess several pharmacological properties and have been shown to be particularly effective against the entry and replication of several viruses. We show that emodin and berberine trigger a virucidal effect on ZIKV. When the virus was exposed to 160 µM of berberine, a reduction of 77.6% in the infectivity was observed; when emodin was used (40 µM), this reduction was approximately 83.3%. Dynamic light scattering data showed that both compounds significantly reduce the hydrodynamic radius of virus particle in solution. We report here that berberine and emodin, two natural compounds, have strong virucidal effect in Zika virus.

Viruses are important agents of emerging zoonoses and are a substantial public health issue. Among emerging viruses, an important group are arboviruses, which are characterized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of vertebrate hosts. Recently, bats have received increasing attention as an important source for the emergence of zoonoses and as possible viral reservoirs. Among the arboviruses, there are many representatives of the genera Flavivirus and Alphavirus, which are responsible for important epidemics such as Dengue virus, Zika virus and Chikungunya virus. Due to the importance of analyzing potential viral reservoirs for zoonosis control and expanding our knowledge of bat viruses, this study aimed to investigate the presence of viruses of the Alphavirus and Flavivirus genera in bats. We analyzed serum, liver, lungs and intestine from 103 bats sampled in northeast and southern Brazil via Nested-PCR and the hemagglutination inhibition test. All samples tested in this study were negative for arboviruses, suggesting that no active or past infection was present in the captured bats. These data indicate that the bats examined herein probably do not constitute a reservoir for these viruses in the studied areas. Further studies are needed to clarify the role of bats as reservoirs and sources of infection of these viral zoonoses., Competing Interests: The authors have declared that no competing interests exist.

In Africa, Old World Primates are involved in the maintenance of sylvatic circulation of ZIKV. However, in Brazil, the hosts for the sylvatic cycle remain unknown. We hypothesized that free-living NHPs might play a role in urban/periurban ZIKV dynamics, thus we undertook an NHP ZIKV investigation in two cities in Brazil. We identified ZIKV-positive NHPs and sequences obtained were phylogenetically related to the American lineage of ZIKV. Additionally, we inoculated four C. penicillata with ZIKV and our results demonstrated that marmosets had a sustained viremia. The natural and experimental infection of NHPs with ZIKV, support the hypothesis that NHPs may be a vertebrate host in the maintainance of ZIKV transmission/circulation in urban tropical settings. Further studies are needed to understand the role they may play in maintaining the urban cycle of the ZIKV and how they may be a conduit in establishing an enzootic transmission cycle in tropical Latin America.

[This corrects the article DOI: 10.1371/journal.pone.0187630.].

Hepatitis C is a disease caused by the hepatitis C virus (HCV), and an estimated 3% of the world population is infected with the virus. During replication, HCV interacts with several cellular proteins. Studies have shown that several heat shock proteins (HSPs) have an altered expression profile in the presence of the virus, and some HSPs interact directly with HCV proteins. In the present study, we evaluated the expression levels of heat shock proteins in vitro in the presence and absence of HCV. The differential expression of 84 HSPs and chaperones was observed using a qPCR array, comparing HCV uninfected and infected Huh7.5 cells. To validate qPCR array, the differentially expressed genes were tested by real-time PCR in three different HCV models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all three viral models. We silenced HSPB8 expression and observed an increase in viral replication. In contrast, when we increased the expression of HSPB8, a decrease in the HCV replication rate was observed. The same procedure was adopted for DNAJC5B, and HCV showed a similar replication pattern as that observed for HSPB8. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and that DNAJC5B has the same function, with more relevant results for genotype 3. We also evaluated the direct interactions between HCV and HSP proteins, and the IP experiments showed that the HCV NS4B protein interacts with HSPB8. These results contribute to a better understanding of the mechanisms involved in HCV replication.

Background: Germline pathogenic variants in BRCA1 and BRCA2 (BRCA) are the main cause of Hereditary Breast and Ovarian Cancer syndrome (HBOC)., Methods: In this study we evaluated the mutational profile and prevalence of BRCA pathogenic/likely pathogenic variants among probands fulfilling the NCCN HBOC testing criteria. We characterized the clinical profile of these individuals and explored the performance of international testing criteria., Results: A pathogenic/likely pathogenic variant was detected in 19.1% of 418 probands, including seven novel frameshift variants. Variants of uncertain significance were found in 5.7% of individuals. We evaluated 50 testing criteria and mutation probability algorithms. There was a significant odds-ratio (OR) for mutation prediction (p ≤ 0.05) for 25 criteria; 14 of these had p ≤ 0.001. Using a cutoff point of four criteria, the sensitivity is 83.8%, and the specificity is 53.5% for being a carrier. The prevalence of pathogenic/likely pathogenic variants for each criterion ranged from 22.1% to 55.6%, and criteria with the highest ORs were those related to triple-negative breast cancer or ovarian cancer., Conclusions: This is the largest study of comprehensive BRCA testing among Brazilians to date, and the first to analyze clinical criteria for genetic testing. Several criteria that are not included in the NCCN achieved a higher predictive value. Identification of the most informative criteria for each population will assist in the development of a rational approach to genetic testing, and will enable the prioritization of high-risk individuals as a first step towards offering testing in low-income countries.

Hepatitis C virus (HCV) is one of the main causes of liver disease and transplantation worldwide. Current therapy is expensive, presents additional side effects and viral resistance has been described. Therefore, studies for developing more efficient antivirals against HCV are needed. Compounds isolated from animal venoms have shown antiviral activity against some viruses such as Dengue virus, Yellow fever virus and Measles virus. In this study, we evaluated the effect of the complex crotoxin (CX) and its subunits crotapotin (CP) and phospholipase A2 (PLA2-CB) isolated from the venom of Crotalus durissus terrificus on HCV life cycle. Huh 7.5 cells were infected with HCVcc JFH-1 strain in the presence or absence of these toxins and virus was titrated by focus formation units assay or by qPCR. Toxins were added to the cells at different time points depending on the stage of virus life cycle to be evaluated. The results showed that treatment with PLA2-CB inhibited HCV entry and replication but no effect on HCV release was observed. CX reduced virus entry and release but not replication. By treating cells with CP, an antiviral effect was observed on HCV release, the only stage inhibited by this compound. Our data demonstrated the multiple antiviral effects of toxins from animal venoms on HCV life cycle.

Hepatitis C virus (HCV) affects about 170 million people worldwide. The current treatment has a high cost and variable response rates according to the virus genotype. Acridones, a group of compounds extracted from natural sources, showed potential antiviral actions against HCV. Thus, this study aimed to evaluate the effect of a panel of 14 synthetic acridones on the HCV life cycle. The compounds were screened using an Huh7.5 cell line stably harbouring the HCV genotype 2a subgenomic replicon SGR-Feo-JFH-1. Cells were incubated in the presence or absence of compounds for 72 h and cell viability and replication levels were assessed by MTT and luciferase assays, respectively. At a concentration of 5 µM the acridone Fac4 exhibited a >90 % inhibition of HCV replication with no effect on cell viability. The effects of Fac4 on virus replication, entry and release steps were evaluated in Huh7.5 cells infected with the JFH-1 isolate of HCV (HCVcc). Fac4 inhibited JFH-1 replication to approximately 70 %, while no effect was observed on virus entry. The antiviral activity of Fac4 was also observed on viral release, with almost 80 % of inhibition. No inhibitory effect was observed against genotype 3 replication. Fac4 was able to intercalate into dsRNA, however did not inhibit NS5B polymerase activity or translation driven by the HCV IRES. Although its mode of action is partly understood, Fac4 presents significant inhibition of HCV replication and can therefore be considered as a candidate for the development of a future anti-HCV treatment.

Introduction: The association between the BTD genotype and biochemical phenotype [profound biotinidase deficiency (BD), partial BD or heterozygous activity] is not always consistent. This study aimed to investigate the genotype-biochemical phenotype association in patients with low biotinidase activity., Methods: All exons, the 5'UTR and the promoter of the BTD gene were sequenced in 72 Brazilian individuals who exhibited low biotinidase activity. For each patient, the expected biochemical phenotype based on the known genotype was compared with the observed biochemical phenotype. Additional non-genetic factors that could affect the biotinidase activity were also analysed., Results: Most individuals were identified by neonatal screening (n = 66/72). When consecutive results for the same patient were compared, age, prematurity and neonatal jaundice appeared to affect the level of biotinidase activity. The biochemical phenotype at the time of the second blood collection changed in 11/22 patients compared to results from the first sample. Three novel variants were found: c.1337T>C (p.L446P), c.1466A>G (p.N489S) and c.962G>A (p.W321*). Some patients with the same genotype presented different biochemical phenotypes. The expected and observed biochemical phenotypes agreed in 68.5% of cases (concordant patients). The non-coding variants c.-183G>A, c.-315A>G and c.-514C>T were present in heterozygosis in 5/17 discordant patients. In addition, c.-183G>A and c.-514C>T were also present in 10/37 concordant patients., Conclusions: The variants found in the promoter region do not appear to have a strong impact on biotinidase activity. Since there is a disparity between the BTD genotype and biochemical phenotype, and biotinidase activity may be affected by both genetic and non-genetic factors, we suggest that the diagnosis of BD should be based on more than one measurement of plasma biotinidase activity. DNA analysis can be of additional relevance to differentiate between partial BD and heterozygosity.

Issues on the correlation of viral genetic diversity and treatment response to the hepatitis C infection remain uncertain. The bottleneck effect dictates the characteristics of the viral population that will establish the infection in a new host and is related to how the immune system and treatment will be effective against the virus. Here we evaluated the phylogenetic characteristics of quasispecies population and the treatment response pattern of a HCV infected couple. We also analyzed whether the viral population of these patients indicated that they were exposed to the same source for primer infection. This study included two patients (P10 and P11) HCV genotype 1b infected. The couple presented horizontal transmission. Viral RNA was isolated from serum samples collected before, during and after treatment, at specific time points. The HCV NS5A gene sequence was amplified, cloned and sequenced. Genetic and evolutionary analyses were performed to compare the quasispecies population of these two patients and local control patients. Genetic distance and diversity were calculated. Phylogenetic analyses were performed by using maximum likelihood and Bayesian methodologies. The analysis of the baseline samples showed that the genetic distance of the viral populations of patients P10 and P11 was significantly lower than when these patients and the control group based on sequences from local patients were analyzed, supporting the horizontal transmission hypothesis. Phylogenetic analysis with sequences from all the time point samples also demonstrated two patterns of evolution depending on the treatment response. The Bayesian analysis showed that one isolate corresponding to the baseline sample of P10 was grouped into the P11 clade, suggesting a way of infection and a bottleneck effect. Our data suggests that the patient P11 viral population may be originated from variants from P10 patient and consequently showing that clinical differences between treatment responses can emerge from the bottleneck effect on viral populations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Culex Flavivirus (CxFV) is an insect-specific virus that is widely distributed and primarily infects mosquito species from the genus Culex. Its hosts include Culex tritaeniorhynchus, Culex quinquefasciatus, and Anopheles sinensis mosquitoes. Since its original identification, CxFV has been reported in several countries. Despite the increasing number of reports on CxFV, little is known about its genomic characteristics. It is unclear whether the phylogenetic relationships between the strains are influenced by host species and geographic location., Results: We characterized the Brazilian CxFV strain and performed a comprehensive genetic and phylogenetic characterization of CxFV based on all ORF sequences described so far. Our results revealed that the Brazilian strain is in a monophyletic clade with the Mexican strain. Overall, selective pressure indicates that the ORF is undergoing purifying selection., Conclusions: The phylogenetic analysis revealed a strong association between climate and CxFV ancestry. Also, based on phylogeny and the genetic distance between the main branches of the tree, we propose the classification of the available sequences into two different genotypes. We also suggest the existence of two different subtypes within Genotype 1.

Germline mutations in BRCA1 or BRCA2 (BRCA) are responsible for 5-15% of breast (BC) and ovarian cancers (OC), predisposing to the development of early onset and often multiple primary tumors. Since mutation carriers can benefit from risk-reducing interventions, the identification of individuals with hereditary breast and ovarian cancer (HBOC) syndrome has a significant clinical impact. We assessed whether a panel assay for recurrent Hispanic BRCA mutations (HISPANEL) has an adequate breadth of coverage to be suitable as a cost effective screening tool for HBOC in a cohort of patients from Southern Brazil. A multiplex, PCR-based panel was used to genotype 232 unrelated patients for 114 germline BRCA mutations, finding deleterious mutations in 3.5% of them. This mutation prevalence is within the range detected by the HISPANEL among BC patients unselected for family history in other Latin American settings. The HISPANEL would have accounted for 27% of the BRCA mutations detected by complete sequencing in a comparison cohort (n = 193). This prevalence may be region-specific since significant differences in population structure exist in Brazil. Comprehensive analysis of BRCA in a larger set of HBOC patients from different Brazilian regions is warranted, and the results could inform customization of the HISPANEL as an affordable mutation screening tool., (Copyright © 2016. Published by Elsevier Inc.)

Ulnar neuropathy at the wrist (UNW) is rare, and always challenging to localize. To increase the sensitivity and specificity of the diagnosis of UNW many authors advocate the stimulation of the ulnar nerve (UN) in the segment of the wrist and palm. The focus of this paper is to present a modified and simplified technique of sensory nerve conduction (SNC) of the UN in the wrist and palm segments and demonstrate the validity of this technique in the study of five cases of type III UNW. The SNC of UN was performed antidromically with fifth finger ring recording electrodes. The UN was stimulated 14 cm proximal to the active electrode (the standard way) and 7 cm proximal to the active electrode. The normal data from amplitude and conduction velocity (CV) ratios between the palm to finger and wrist to finger segments were obtained. Normal amplitude ratio was 1.4 to 0.76. Normal CV ratio was 0.8 to 1.23.We found evidences of abnormal SNAP amplitude ratio or substantial slowing of UN sensory fibers across the wrist in 5 of the 5 patients with electrophysiological-definite type III UNW.

Knowledge of placebo and nocebo effects is essential to identify their influence on the results in clinical practice and clinical trials, and thereby properly interpret their results. It is known that the gold standard of clinical trials research is the double-blind, placebo-controlled, randomized clinical study. The objective of this review is to distinguish specific from non-specific effects, so that the presence of positive effects in the group that received placebo (placebo effect) and the presence of adverse effects in the group receiving placebo (nocebo effect) lead to confounding in interpreting the results. Placebo and nocebo effects have been considered in neurological diseases such as depression, pain, headache, multiple sclerosis, epilepsy. As placebo and nocebo effects are also present in clinical practice, the purpose of this review is to draw attention to their influence on neurological practice, calling attention to the development of measures that can minimize them.

Neurofibromatosis type 1 (NF1) patients are more likely to have vitamin D deficiency when compared to the general population. This study aimed to determine the levels of 25-OH-vitamin D [25(OH)D] in individuals with NF1 and disease-unaffected controls and analyze FokI and BsmI VDR gene polymorphisms in a case and in a control group. Vitamin D levels were compared between a group of 45 NF1 patients from Southern Brazil and 45 healthy controls matched by sex, skin type, and age. Genotypic and allelic frequencies of VDR gene polymorphisms were obtained from the same NF1 patients and 150 healthy controls. 25(OH)D deficiency or insufficiency was not more frequent in NF1 patients than in controls (p = 0.074). We also did not observe an association between FokI and BsmI VDR gene polymorphisms and vitamin D levels in NF1 patients, suggesting that their deficient or insufficient biochemical phenotypes are not associated with these genetic variants. The differences between the groups in genotypic and allelic frequencies for FokI and BsmI VDR gene polymorphisms were small and did not reach statistical significance. These polymorphisms are in partial linkage disequilibrium and the haplotype frequencies also did not differ in a significant way between the two groups (p = 0.613).

Introduction: No published clinical trial data are available to inform the use of enzyme replacement therapy (ERT) in patients with the severe (neuropathic) phenotype of mucopolysaccharidosis II (MPS II). Current guidelines recommend ERT administered intravenously be used on a trial basis in this population., Aims/methods: A retrospective chart review was conducted at five international centers for this case series of 22 patients with neuropathic MPS II who received intravenous idursulfase 0.5 mg/kg weekly for at least 2 consecutive years. We collected data about urinary glycosaminoglycan levels, adverse events, and the following somatic signs/symptoms: skeletal disease, joint range of motion, liver/spleen size, respiratory infections, cardiac disease, diarrhea, skin/hair texture, and hospitalizations., Results: The age at diagnosis was 2 months to 5 years, and the age at idursulfase initiation was between 18 months and 21 years. One of 22 patients experienced improvements in seven somatic signs/symptoms; 17/22 experienced improvements in five to six somatic signs/symptoms; and 4/22 experienced improvements in four somatic signs/symptoms. None experienced fewer than four improvements. No new safety concerns arose. Infusion-related reactions were experienced by 4/22 patients but were successfully managed using accepted strategies., Conclusions: Long-term treatment with idursulfase was associated with improvements in somatic manifestations in this case series of patients with neuropathic MPS II. The family and medical team should maintain open lines of communication to make treatment decisions that take into consideration the benefits and limitations of ERT in this population.