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Introduction: The disease caused by the new Coronavirus (SARS-CoV-2) was called COVID-19 and has currently been a public and emergency health concern in Brazil and other countries. Aim: This article aims to describe the statistical associations of a group of patients who progressed to death with COVID-19 and who had a low level of Vitamin D in the blood. Methods: This is an observational, case-control and clinical study involving 103 patients with COVID-19 and with severe symptoms that progressed to death. Results: A total of 92 patients (89.3%) infected with COVID-19 died and had serum vitamin D levels significantly lower than 30 ng/ml. However, a total of 80 (77.6%) patients had a Vitamin D level of less than 20 ng/ml. Compared with the control group, all-cell levels of inflammatory markers were significantly higher in blood serum when level with Vitamin D from COVID-19 patients in the treatment group (n ≤ 30 ng/ml). Conclusion: This study showed that patients with serum Vitamin D deficiency are more susceptible to the worsening of COVID-19 and it is generally associated with the release of cytokines as cellular markers mainly in the increase in pro-inflammatory cytokines.

The Spike protein's structure of the SARS-CoV-2 provides a unique opportunity to consider perturbations at the atomic level. We used the cryo-electron microscopy structure of the open conformation of the Spike protein to assess the impact of the mutations observed in the variants of concern at the molecular level. Molecular dynamics were subsequently performed with both the wt and the mutated forms to compare the flexibility and variation data for each residue of the three-dimensional fluctuations in the region associated with each alpha carbon. Additionally, protein-protein docking was used to investigate the interaction of each mutated profile with the ACE-2 receptor. After the molecular dynamics, the results show that the mutations increased the stability of the trimeric protein, with greater stability observed in the Gamma variant harboring the 10 characteristic mutations. The results of molecular dynamics, as shown by RMSF demonstrated for the residues that comprise the binding domain receptor (RBD), exhibited a reduction in flexibility, which was more pronounced in the Gamma variant. Finally, protein-protein docking experiments revealed an increase in the number of hydrophobic interactions and hydrogen bonds in the Gamma variant against the ACE-2 receptor, as opposed to the other variants. Taken together, these

Objective Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.

Chikungunya virus (CHIKV) is an arbovirus ( Togaviridae family, Alphavirus genus), first identified in 1953 in Tanzania. In 2005, CHIKV emerged in India, and later caused outbreaks in Southeast Asia, Oceania and the Americas. Some Clinical signs are associated with CHIKV infection include fever and/or concomitant arthralgia, neurological manifestations and death. However, in infections caused by other arboviruses, such as the Dengue virus and West Nile virus, its often observed changes in liver enzymes. This study aims to evaluate the profile of the biochemical markers for kidney and liver injury patients infected with CHIKV in acute phase of infection. We found a significant elevation on the levels of creatinine in CHIKV-infected people, possibly associated with myalgia and indicative of muscle damage. The novelty was the elevated levels of creatinine found during a long period.

Spike (S) protein has been recognized as a promising molecular target for diagnostic, vaccines and antiviral drugs development for COVID-19. In this study, we analyzed the most predominant mutations in the S protein of Brazilian isolates and predicted the effect of these amino acid alterations to protein conformation. A total of 25,924 sequences were obtained from GISAID for five regions of Brazilian territory (Midwest, North, Northeast, South, and Southeast), according to exclusion criteria. Most of the SARS-CoV-2 isolates belongs to the G clade and showed a large occurrence of D614G, N501Y and L18F substitutions. Prediction effects of these amino acid substitutions on the structure dynamics of the spike protein indicated a positive ΔΔG values and negative ΔΔSVib in most cases which is associated to structural stabilization and flexibility reduction of the S protein. Mutations E484K, N501Y and K417N belong to several SARS-CoV-2 variants of concern such as Alpha, Beta, Gamma and Delta, and showed high incidence among Brazilian isolates. These mutations have been described to increase RBD affinity to ACE-2 host and abolishment of RBD affinity to potent neutralizing ant-RBD. The increase in rates of infection and reinfection requires continuous genomic surveillance studies in order to characterize emerging mutations and monitor vaccine efficacy, and thus consideration structural data and dynamics in the observed phenotypes.Communicated by Ramaswamy H. Sarma.

The urea- and pressure-induced dissociation of tobacco mosaic virus (TMV) and proton involvement were investigated using a thermodynamic approach that considered the extent of reaction for protons and urea. The stoichiometry of these effectors was determined, as were the Gibbs free energy of dissociation (ΔGD), the volume change associated with dissociation (ΔV) and the Hill coefficient (H). At low pressure, the proton release ($$\mu_{{H^{ + } }}$$) was 1.9 and 1.4 mol H+/mol of TMV at pH 4.8 and 6.8, respectively. Below pH 4.5 and at pH 6.2, there was proton absorption ($$\mu_{{H^{ + } }}$$

Cytosolic phospholipase A2 (cPLA2) belongs to a large family of proteins and plays a crucial role in the regulation of arachidonic acid metabolism and inflammation cascade in zebrafish (Danio rerio). This enzyme with a molecular weight of 85 kDa, has two distinct domains. One is the regulatory and calcium-dependent (Ca

A computational procedure based on the numerical integration of the Michaelis‐Menten model of enzyme action, free of any restrictions of steady‐state conditions and substrate/enzyme ratios is proposed. The original Michaelis‐Menten data for invertase (Michaelis and Menten, 1913, Biochem Z. 49:333‐369) were reanalyzed. The surface and contour plots that were generated for substrate, free enzyme, complex, and product confirmed the model's usefulness. All energy potentials G and the “conformational drift parameter” δ involved in the enzymatic reactions were determined. Our findings indicate that at so = 0.0052 M the enzyme‐substrate (ES) complex present an energy of dissociation of GE + S➔ES = 15.0 kJ/mol and as so increases to 0.333 M, the GE + S➔ES value decreases to 5.0 kJ/mol, thereby decreasing its presence in solution. Overall, the ability to determine G and δ for each transition suggests a relationship between kinetics and thermodynamics. The analysis proposed here can be directly applied to chemical and biological situations, as well as industrial processes.

Pediatric adrenocortical tumors (pACT) are rare, display complex genomic background and lack robust prognostic markers. Very recently, distinct genomic methylation profiles of pACT were associated with prognosis. The vitamin D receptor (VDR) was shown to be underexpressed in ACT, especially in carcinomas (ACC). In adult ACC, VDR inactivation by methylation was demonstrated. On the other hand, VDR activation was shown to inhibit ACC proliferation in vitro and in vivo. Aim: To evaluate VDR DNA methylation profile and its clinical and prognostic significance in pediatric ACT. Methods: Genomic DNA methylation from 57 pACTs [40 girls; median age: 2.1 (0.2-16.4) years] was assessed using Infininium Methylation EPIC BeadChip Array. Unsupervised hierarchical clustering analysis (Ward method, R Stats Package) was performed considering the M-values of the 49 probes targeting the whole extension of VDR gene contained in the array. Clinical, histopathological and molecular features, as well as pACT VDR mRNA levels (qPCR) and nuclear immunoreactivity (IHC) were used for association analysis. Results: Hierarchical clustering identified three clusters of pACT. Methylated VDR-targeted probes (M-values different from 0; n=37) composed the VDR methylation profile, which differed significantly between the clusters [M-values: C1=1.77 (1.1-1.9) (low), C2=2.15 (1.7-2.7) (intermediate), and C3=2.65 (2.2-3.1) (high); p=4), were not carriers of somatic Beta-catenin activating mutations, or died. Although cluster C2 patients (n=21) presented intermediary disease features, only 2 patients died and the overall outcome was positive. Instead, the C3 cluster concentrated patients (n=18) with non-localized/metastatic disease (IPACTR stages I/II vs. III/IV; p=0.004), post-surgical metastasis/recurrence (p=0.009), and patients who needed adjuvant chemotherapy (p=0.005). Moreover, C3 patients had lower overall and disease-free survival rates (log-rank: p=0.001 and p=0.014, respectively). VDR methylation was not associated with sex, clinical presentation, P53 mutations, nor with tumor VDR mRNA expression or nuclear immunoreactivity. Conclusions: Three VDR methylation profiles were associated with distinct pACT clinical features and outcome. High VDR methylation was associated with worst outcome. Fully functioning VDR may play a beneficial role against pediatric adrenocortical tumorigenesis. This finding highlights the potential of targeting VDR as an adjuvant therapeutic target.

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1h) at −18 °C, compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set.In silicoprediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The approach used here provided important information on the antibody/antigen interactions required to improve B cell responses to PPV and may be useful in developing novel strategies for producing new vaccines.Abbreviations3D, three dimensional; HHP, high hydrostatic pressure; ID, identification; IEDB, Immune Epitope Database; ORFs, open reading frames; p.i., post-infection; PPV, porcine parvovirus; RMSD, root-mean-square deviation of atomic positions; R(g), radius of gyration. RMSF, root-mean-square fluctuation; SK6, Swine kidney cell; SPF, specific pathogen free; TCID50/mL, Median Tissue Culture Infectious Dose; HI, Hemagglutination Inhibition.

Chikungunya virus (CHIKV) is a re-emerging arbovirus that is causing outbreaks in several countries of the Americas. The virus was introduced in Brazil in 2014, and since then, several Brazilian states have notified autochthonous cases.Provide additional evidence on a CHIKV outbreak and an outline of the laboratory and clinical profile of symptomatic patients in Sergipe, Brazil.In February 2016, we collected 142 serum samples from symptomatic patients for arboviruses in Sergipe, Brazil. All samples were submitted to qRT-PCR for the emerging arboviruses circulating in Brazil - ZIKV, CHIKV, and DENV - and later submitted to the immunoenzymatic assay. RNA positive samples were randomly selected and sequenced for characterization of the genotype involved in the outbreak.Our study had 75.35% (107/142) positivity for CHIKV infection, with all age groups and genera being equally infected. The virus was identified in 11 of the 13 cities studied in that state, including the ECSA genotype. Importantly, fever was the only statistically significant symptoms for CHIKV infection (p0.05), while asthenia was significantly associated with symptomatic patients that were CHIKV-negative (p0.05).Our findings support the importance of fever as a clinical marker and contribute to molecular and serological surveillance data, which may help in the understanding of CHIKV circulation, emergence and clinical description.

The Zika virus (ZIKV) arrival in Brazilian territory brought to light the need for preparedness regarding arboviruses in Brazil. Compound screening is a cumbersome process dependent upon in vitro testing and validation. Recently, virtual screening methods have improved precision and reliability providing a framework for in silico testing of lead compound candidates. Here we have applied these methods on compounds that were previously shown to be active against Dengue virus in vitro, taking the structural information of such compounds and applying docking methods to identify putative binding sites. A molecular dynamics approach was also used to refine the docking results. The computational experiments ran here suggests that compounds such as Epigallocatechin Gallate, Ergotamine and Avermectin-B1a bind to active sites on the viral enzymes NS5 and NS3, as well as on its Envelope protein. Refinement shows that such bindings were not lost during the production run and key regions on both enzymes were structurally displaced on average over the simulation time. Interestingly there is no documented drug interactions among these candidates, raising the possibility of drug combinations during treatments. Moreover, the candidate compounds have been extensively studied, thus providing important information regarding intracellular interactions caused by them, which are also associated with pathways exploited by the virus, suggesting possible side interactions hindering the replication process.

BACKGROUND Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

To the Editor: Tadarida brasiliensis (I. Geoffroy, 1824) is a species of free-tailed bat that has resident and migratory populations in Brazil (1). This species has adapted to urban areas, occupying roofs, ceilings, and other human constructions, and often coexists with other bat species and humans (2), enabling epidemiologic risks (3). In recent studies, an alphacoronavirus has been detected in urban bat species Molossus molossus, M. rufus, and Tadarida brasiliensi in Brazil (4,5). Evidence suggests that alphacoronaviruses may use bats as hosts to spread human coronavirus (HCoV) NL63, which originated by evolution of Appalachian Ridge CoV strain 2 (ARCoV.2) (6). In this study, a total of 20 anal and tracheal swab samples from 10 bats (T. brasiliensis) were collected at the Jequitibas Wood, in Campinas, Sao Paulo State, Brazil (22°54′31.34′′S 47°02′58.01′′W). We extracted viral genetic material using the RNA Extraction Mini Kit (QIAGEN, Hilden, Germany) and synthesized cDNA using random primers from the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s protocol. Samples were analyzed by conventional reverse transcription PCR assays using panCoV primers targeting a 215-bp replicase fragment as previously described (7) with slight modifications to include more cycles and less extension time in order to obtain more PCR products. Sequencing reactions on a Pancoronavirus-positive anal swab sample (7) were performed at Central Laboratory of High Performance Technologies in Life Sciences (LaCTAD) at UNICAMP (http://www.lactad.unicamp.br) using an automated sequencer (3730xl DNA Analyzer; Applied Biosystems). The chromatograms were edited using the program UGENE version 1.14 (UGENE, http://ugene.unipro.ru/forum/YaBB.pl?num=1407749393) and evaluated using Phred scores for base calling. Alignment was made with ClustalW v.2.1 software (http://www.clustal.org) implemented on Linux command line interface, and a similarity matrix was generated with sequences retrieved from the GenBank database. A 144-nt fragment of the replicase gene was obtained after editing, and phylogenetic analysis was performed after determining the best evolution model by using the jModelTest2 software (https://code.google.com/p/jmodeltest2/). Different CoV sequences were included to represent the genera Alpha-, Beta-, Gamma-, and Deltacoronavirus. Clustering with the ARCoV.2 and porcine epidemic diarrhea virus (PEDV) was obtained using the maximum-likelihood (ML) method after 1,000 Shimodaira-Hasegawa–like support values with the general time-reversible model and category approximation in 20 rates category in a gamma distribution (Technical Appendix Figure, panel A) and neighbor-joining methods under Kimura-2-parameter and 1,000 replicates of bootstrap (Technical Appendix Figure, panel B). Subsequently, metagenomic analysis was made by creating a pool of the 10 bat samples. Samples were resuspended in Dulbecco Modified Eagle Medium (Life Technologies-GIBCO, Grand Island, NY, USA) and filtered through 0.22 μm. The recovered sample was then treated with DNase (Invitrogen, Carlsbad, CA, USA) to remove contaminating DNA and with Proteinase K (Invitrogen) to eliminate inhibitors and to disrupt viral capsids. Samples were then subjected to RNA extraction (QIAGEN) and sent to the sequencing core facility. Sequencing was performed on Illumina HiSeq2500 instrument by using the 2×100 bp kit according to manufacturer’s instructions. Through these analyses, we obtained 34,409,110 reads, of which 76.47% had quality index ≥30. The contigs were assembled by de novo genome assembly (blastx E-value ≤1–5) (8) generating 10.742 scaffolds: 35 matches for coronaviruses (using the Coronavirus Database, http://covdb.microbiology.hku.hk), 3 matches for PEDV, and 2 matches for HCoV-NL63 (both using the UniProt database, http://www.uniprot.org) (Technical Appendix). The sequences obtained had 87.5% (126/144) nucleotide identity with ARCoV.2, an unclassified alphacoronavirus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JX537912","term_id":"407016953","term_text":"JX537912"}}JX537912) for which a zoonotic role has been suggested (6). Preliminary analysis indicated good coverage of the polymerase region of the ARCoV.2 reference sequence by the reads (quality index >40) by using reference assembly against CoV complete genomes. This finding reinforces the hypothesis of this viral agent in the specimens analyzed. Moreover, molecular assays are underway in our laboratory to elucidate the alternative hypothesis of PEDV presence in bats in Brazil. In summary, we found that a CoV detected in T. brasiliensis bats in Brazil has close phylogenetic relationships to ARCoV.2 and PEDV. Considering the zoonotic impact of these viral agents on the emergence of new diseases in animal and human populations, we believe that both results may strongly contribute to a better understanding of the molecular eco-epidemiology of these alphacoronaviruses. The reconstruction of their evolutionary history to trace their occurrence in humans and in bat populations as well as in other animals is being conducted to clarify their evolutionary pathway. Technical Appendix: Maximum likelihood and neighbor-joining genetic sequences of coronaviruses detected in bats of Tadarida brasiliensis species. Click here to view.(57K, pdf)

A duplex reverse transcription polymerase chain reaction (RT-PCR) and direct immunofluorescence assays (DFAs) were evaluated for detection of influenza types A and B in comparison with virus isolation in Madin-Darbin canine kidney cells. Four hundred four nasal wash were collected from individuals presenting with acute respiratory symptoms during 2001 to 2003 influenza seasons. According to the reference method, 78 (19.3%) samples were infected by influenza virus: 46 were type A and 32 type B. The overall concordance between the 3 assays was 96%, with 317 negative and 71 positive samples in all tests. RT-PCR reached 92.3% sensitivity and 98.5% specificity, and for DFA, the corresponding values were 93.6% and 97.2%, respectively. DFA and RT-PCR could be applied in different routine settings, resulting as an advantage compared with virus isolation: DFA provides rapid results for clinical purposes, but RT-PCR allows running more samples, an important concern in early pandemic circumstances.

Avian metapneumovirus (aMPV) is a negative-sense single-stranded RNA enveloped virus of the Metapneumovirus genus belonging to theParamyxoviridae family. This virus may cause significant economic losses to the poultry industry, despite vaccination, which is the main tool for controlling and preventing aMPV. The aim of this study was to evaluate the antiviral activity of extracts of four different native plants of the Brazilian Cerrado against aMPV. The antiviral activity against aMPV was determined by titration. This technique measures the ability of plant extract dilutions (25 to 2.5 µg mL-1) to inhibit the cytopathic effect (CPE) of the virus, expressed as inhibition percentage (IP). The maximum nontoxic concentration (MNTC) of the extracts used in antiviral assay was 25 µg mL-1for Aspidosperma tomentosumand Gaylussacia brasiliensis, and 2.5 µg mL-1for Arrabidaea chicaand Virola sebifera. Twelve different extracts derived from four plant species collected from the Brazilian Cerrado were screened for antiviral activity against aMPV. G. brasiliensis, A. chica,and V. sebifera extracts presented inhibition rates of 99% in the early viral replication stages, suggesting that these extracts act during the adsorption phase. On the other hand, A. tomentosum inhibited 99% virus replication after the virus entered the cell. The biomonitored fractioning of extracts active against aMPV may be a tool to identify the active compounds of plant extracts and to determine their precise mode of action.

The immune system is a network of thousands of molecules, cells and regulatory factors that produce many interrelated responses. In this study, we used spot synthesis to map tobacco mosaic virus (TMV) epitopes in a mice animal model (Balb/c) and compared the results with those obtained using immunoinformatic prediction tools. Mice were inoculated with TMV and after immunization the sera were incubated with an array of overlapping pentadecapeptides that corresponded to the full sequence of the TMV capside protein (TMVcp) that had been synthesized on a cellulose membrane for spot synthesis analysis. Six linear epitopes were identified experimentally, as shown by the IgG-elicited immune responses in mice. The data for epitope prediction based on epitope databases agreed with the results obtained by spot synthesis results. Comparison of the findings for spot intensities and those obtained with the prediction software allowed the identification of different responses according to the MHC class I alleles. The results of this work provide a detailed antigenic profile for TMV.