Genomic characterization and molecular evolution of human monkeypox viruses.

Monkeypox virus is a member of the family Poxviridae, as are variola virus and vaccinia virus. It has a linear double-strand DNA genome approximately 197 kb long, containing ~190 non-overlapping ORFs. Comparison of members of the Central and West African clades shows the presence of unique genes that are associated with different disease presentations, depending on the strain. The last smallpox vaccination efforts ended in the mid-1980s, and there is concern about the recent spread of human monkeypox disease around the world. Almost 87,000 human monkeypox cases have been diagnosed in the world, of which more than 10,900 were in Brazil. The aim of this study was to evaluate the epidemiology and molecular evolution of hMpxV. From computational biology analysis of 640 hMpxV genomes from 1962 to 2022, synteny breaks and gene conservation were observed between Central and West clade genomes, and strains belonged with the 2022 outbreak assigned to the West African clade. Evidence was found for diversifying selective pressure at specific sites within protein coding sequences, acting on immunomodulatory processes. The existence of different sites under diversifying and purifying selection in paralog genes indicates adaptive mechanisms underlying the host-pathogen interaction of monkeypox virus in humans.

Population-based prevalence surveys during the Covid-19 pandemic: A systematic review.

Population-based prevalence surveys of Covid-19 contribute to establish the burden of infection, the role of asymptomatic and mild infections in transmission, and allow more precise decisions about reopen policies. We performed a systematic review to evaluate qualitative aspects of these studies, assessing their reliability and compiling practices that can influence the methodological quality. We searched MEDLINE, EMBASE, bioRxiv and medRxiv, and included cross-sectional studies using molecular and/or serological tests to estimate the prevalence of Covid-19 in the general population. Survey quality was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Prevalence Studies. A correspondence analysis correlated methodological parameters of each study to identify patterns related to higher, intermediate and lower risks of bias. The available data described 37 surveys from 19 countries. The majority were from Europe and America, used antibody testing, and reached highly heterogeneous sample sizes and prevalence estimates. Minority communities were disproportionately affected by Covid-19. Important risk of bias was detected in four domains: sample size, data analysis with sufficient coverage, measurements in standard way and response rate. The correspondence analysis showed few consistent patterns for high risk of bias. Intermediate risk of bias was related to American and European studies, municipal and regional initiatives, blood samples and prevalence >1%. Low risk of bias was related to Asian studies, nationwide initiatives, reverse-transcriptase polymerase chain reaction tests and prevalence <1%. We identified methodological standards applied worldwide in Covid-19 prevalence surveys, which may assist researchers with the planning, execution and reporting of future population-based surveys.

Systemic redox imbalance in severe COVID-19 patients.

The aim of this study was to evaluate the systemic redox state and inflammatory markers in intensive care unit (ICU) or non-ICU severe COVID-19 patients during the hospitalization period. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients), 5-7 days after admission (T2: 5-7 days after hospital admission), and at the discharge time from the hospital (T3: 0-72 h before leaving hospital or death) to analyze systemic oxidative stress markers and inflammatory variables. The reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were analyzed in peripheral granulocytes and monocytes. THP-1 human monocytic cell line was incubated with plasma from non-ICU and ICU COVID-19 patients and cell viability and apoptosis rate were analyzed. Higher total antioxidant capacity, protein oxidation, lipid peroxidation, and IL-6 at hospital admission were identified in both non-ICU and ICU COVID-19 patients. ICU COVID-19 patients presented increased C-reactive protein, ROS levels, and protein oxidation over hospitalization period compared to non-ICU patients, despite increased antioxidant status. Granulocytes and monocytes of non-ICU and ICU COVID-19 patients presented lower MMP and higher ROS production compared to the healthy controls, with the highest values found in ICU COVID-19 group. Finally, the incubation of THP-1 cells with plasma acquired from ICU COVID-19 patients at T3 hospitalization period decreased cell viability and apoptosis rate. In conclusion, disturbance in redox state is a hallmark of severe COVID-19 and is associated with cell damage and death.

Evolutionary and structural aspects of Solanaceae RNases T2.

Plant RNases T2 are involved in several physiological and developmental processes, including inorganic phosphate starvation, senescence, wounding, defense against pathogens, and the self-incompatibility system. Solanaceae RNases form three main clades, one composed exclusively of S-RNases and two that include S-like RNases. We identified several positively selected amino acids located in highly flexible regions of these molecules, mainly close to the B1 and B2 substrate-binding sites in S-like RNases and the hypervariable regions of S-RNases. These differences between S- and S-like RNases in the flexibility of amino acids in substrate-binding regions are essential to understand the RNA-binding process. For example, in the S-like RNase NT, two positively selected amino acid residues (Tyr156 and Asn134) are located at the most flexible sites on the molecular surface. RNase NT is induced in response to tobacco mosaic virus infection; these sites may thus be regions of interaction with pathogen proteins or viral RNA. Differential selective pressures acting on plant ribonucleases have increased amino acid variability and, consequently, structural differences within and among S-like RNases and S-RNases that seem to be essential for these proteins play different functions.

Genomic epidemiology of SARS-CoV-2 in Esteio, Rio Grande do Sul, Brazil.

BACKGROUND: Brazil is the third country most affected by Coronavirus disease-2019 (COVID-19), but viral evolution in municipality resolution is still poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We aimed to track molecular evolution and spread of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Esteio (Southern Brazil) using phylogenetics and phylodynamics inferences from 21 new genomes in global and regional context. Importantly, the case fatality rate (CFR) in Esteio (3.26%) is slightly higher compared to the Rio Grande do Sul (RS) state (2.56%) and the entire Brazil (2.74%). RESULTS: We provided a comprehensive view of mutations from a representative sampling from May to October 2020, highlighting two frequent mutations in spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in spike Receptor Binding Domain (RBD) characteristic of the B.1.351 and P.1 lineages, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). E484K was found in two genomes from mid-October, which is the earliest description of this mutation in Southern Brazil. Lineages containing this substitution must be subject of intense surveillance due to its association with immune evasion. We also found two epidemiologically-related clusters, including one from patients of the same neighborhood. Phylogenetics and phylodynamics analysis demonstrates multiple introductions of the Brazilian most prevalent lineages (B.1.1.33 and B.1.1.248) and the establishment of Brazilian lineages ignited from the Southeast to other Brazilian regions. CONCLUSIONS: Our data show the value of correlating clinical, epidemiological and genomic information for the understanding of viral evolution and its spatial distribution over time. This is of paramount importance to better inform policy making strategies to fight COVID-19.

Cestode strobilation: prediction of developmental genes and pathways.

BACKGROUND: Cestoda is a class of endoparasitic worms in the flatworm phylum (Platyhelminthes). During the course of their evolution cestodes have evolved some interesting aspects, such as their increased reproductive capacity. In this sense, they have serial repetition of their reproductive organs in the adult stage, which is often associated with external segmentation in a developmental process called strobilation. However, the molecular basis of strobilation is poorly understood. To assess this issue, an evolutionary comparative study among strobilated and non-strobilated flatworm species was conducted to identify genes and proteins related to the strobilation process. RESULTS: We compared the genomic content of 10 parasitic platyhelminth species; five from cestode species, representing strobilated parasitic platyhelminths, and five from trematode species, representing non-strobilated parasitic platyhelminths. This dataset was used to identify 1813 genes with orthologues that are present in all cestode (strobilated) species, but absent from at least one trematode (non-strobilated) species. Development-related genes, along with genes of unknown function (UF), were then selected based on their transcriptional profiles, resulting in a total of 34 genes that were differentially expressed between the larval (pre-strobilation) and adult (strobilated) stages in at least one cestode species. These 34 genes were then assumed to be strobilation related; they included 12 encoding proteins of known function, with 6 related to the Wnt, TGF-ß/BMP, or G-protein coupled receptor signaling pathways; and 22 encoding UF proteins. In order to assign function to at least some of the UF genes/proteins, a global gene co-expression analysis was performed for the cestode species Echinococcus multilocularis. This resulted in eight UF genes/proteins being predicted as related to developmental, reproductive, vesicle transport, or signaling processes. CONCLUSIONS: Overall, the described in silico data provided evidence of the involvement of 34 genes/proteins and at least 3 developmental pathways in the cestode strobilation process. These results highlight on the molecular mechanisms and evolution of the cestode strobilation process, and point to several interesting proteins as potential developmental markers and/or targets for the development of novel antihelminthic drugs.

Molecular evolution of Pr1 proteases depicts ongoing diversification in Metarhizium spp.

The Pr1 family of serine endopeptidases plays an important role in pathogenicity and virulence of entomopathogens such as Metarhizium anisopliae (Ascomycota: Hypocreales). These virulence factors allow for the penetration of the host cuticle, a vital step in the infective process of this fungus, which possesses 11 Pr1 isoforms (Pr1A through Pr1K). The family is divided into two classes with Class II (proteinase K-like) comprising 10 isoforms further split into three subfamilies. It is believed that these isoforms act synergistically and with other virulence factors, allowing pathogenicity to multiple hosts. As virulence coevolves through reciprocal selection with hosts, positive selection may lead to the evolution of new protease families or isoforms of extant ones that can withstand host defenses. This work tests this hypothesis in Class II Pr1 proteins, focusing on M. anisopliae, employing different methods for phylogenetic inference in amino acid and nucleotide datasets in multiple arrangements for Metarhizium spp. and related species. Phylogenies depict groups that match the taxonomy of their respective organisms with high statistical support, with minor discrepancies. Positively selected sites were identified in six out of ten Pr1 isoforms, most of them located in the proteolytic domain and spatially close to the catalytic residues. Moreover, there was evidence of functional divergence in the majority of pairwise comparisons. These results imply the existence of differential selective pressure acting on Pr1 proteins and a potential new isoform, likely affecting host specificities, virulence, or even adapting the organism to different host-independent lifestyles.

Prion protein conversion triggered by acidic condition: a molecular dynamics study through different force fields.

Prions are proteins that cause a group of invariably fatal neurodegenerative diseases, one of the most known being bovine spongiform encephalopathy. The three-dimensional structure of PrPSc , the altered isoform of the prion protein, has not been fully elucidated yet, and studies on prion conversion mechanisms must rely on hypothetical ß-rich structures. Experimental and computational studies indicate that the use of low pH is capable to produce a gain of ß-structure content in the otherwise unstructured N-terminal region. These in silico studies have used different PrP fragments from distinct organisms, and with different lengths and simulation protocols, making it difficult to identify the influence of the force fields on the formation of such structures. Here, we performed a systematic study of the influence of six well-established force fields (GROMOS96 53a6, GROMOS96 43a1, AMBER99SB, AMBER99SB-ILDN, CHARMM27, and OPLS-AA/L) on the process of structural conversion of the Syrian hamster cellular prion protein simulated at acidic and neutral pH. From our analysis, we observe a strong dependence of the results with the different force fields employed. Additionally, only GROMOS96 53A6 and AMBER99SB force fields are capable to capture a high ß-sheet formation at acidic pH and adequately reproduce the neutral pH. In both cases, the ß-sheet elongation seems to be guided by the movement of the N-terminal tail toward the N-terminal of α-helix HB under acidic condition. These results comprise the most wide-ranging study to date correlating force fields to structural changes in the cellular prion protein. © 2018 Wiley Periodicals, Inc.

Molecular evolution and transcriptional profile of GH3 and GH20 ß-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of ß-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

Contribution of WUSCHEL-related homeobox (WOX) genes to identify the phylogenetic relationships among Petunia species.

Developmental genes are believed to contribute to major changes during plant evolution, from infrageneric to higher levels. Due to their putative high sequence conservation, developmental genes are rarely used as molecular markers, and few studies including these sequences at low taxonomic levels exist. WUSCHEL-related homeobox genes (WOX) are transcription factors exclusively present in plants and are involved in developmental processes. In this study, we characterized the infrageneric genetic variation of Petunia WOX genes. We obtained phylogenetic relationships consistent with other phylogenies based on nuclear markers, but with higher statistical support, resolution in terminals, and compatibility with flower morphological changes.

Diversification and distinctive structural features of S-RNase alleles in the genus Solanum.

The multigenic and multiallelic S-locus in plants is responsible for the gametophytic self-incompatibility system, which is important to prevent the detrimental effects of self-fertilization and inbreeding depression. Several studies have discussed the importance of punctual mutations, recombination, and natural selection in the generation of allelic diversity in the S-locus. However, there has been no wide-ranging study correlating the molecular evolution and structural aspects of the corresponding proteins in Solanum. Therefore, we evaluated the molecular evolution of one gene in this locus and generated a statistically well-supported phylogenetic tree, as well as evidence of positive selection, helping us to understand the diversification of S alleles in Solanum. The three-dimensional structures of some of the proteins corresponding to the major clusters of the phylogenetic tree were constructed and subsequently submitted to molecular dynamics to stabilize the folding and obtain the native structure. The positively selected amino acid residues were predominantly located in the hyper variable regions and on the surface of the protein, which appears to be fundamental for allele specificity. One of the positively selected residues was identified adjacent to a conserved strand that is crucial for enzymatic catalysis. Additionally, we have shown significant differences in the electrostatic potential among the predicted molecular surfaces in S-RNases. The structural results indicate that local changes in the three-dimensional structure are present in some regions of the molecule, although the general structure seems to be conserved. No previous study has described such structural variations in S-RNases.

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

BACKGROUND: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. RESULTS: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. CONCLUSIONS: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

Intense P.1 (Gamma) diversification followed by rapid Delta substitution in Southern Brazil: a SARS-CoV-2 genomic epidemiology study.

The analyses of genetic traits, dispersion patterns and phylogenomics are essential for understanding the evolutionary forces driving SARS-CoV-2 viruses in these three years of COVID-19 pandemics. Brazil is one of the most affected countries in the world and not sufficient genomic studies have been performed. The emergence of P.1 lineage led to one of the most serious public health crises on record. Our study presents the genomic sequencing and characterization of 412 samples from Rio Grande do Sul state, in the Brazilian Southern region, during Gamma and Delta epidemic waves, in 2021. Additionally, molecular evolution tests were performed to identify positively selected sites in Brazil between 2020 and 2022, as well as offer some evolutionary perspective about the maintenance of multiple spike mutations in Omicron lineages. Genomic epidemiology analysis has indicated an intense P.1 (Gamma) diversification followed by rapid Delta substitution in Southern Brazil.

New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis.

BACKGROUND: Mycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts. RESULTS: The overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species. CONCLUSIONS: The comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host's immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.

The MetaGens algorithm for metagenomic database lossy compression and subject alignment.

The advancement of genetic sequencing techniques led to the production of a large volume of data. The extraction of genetic material from a sample is one of the early steps of the metagenomic study. With the evolution of the processes, the analysis of the sequenced data allowed the discovery of etiological agents and, by corollary, the diagnosis of infections. One of the biggest challenges of the technique is the huge volume of data generated with each new technology developed. To introduce an algorithm that may reduce the data volume, allowing faster DNA matching with the reference databases. Using techniques like lossy compression and substitution matrix, it is possible to match nucleotide sequences without losing the subject. This lossy compression explores the nature of DNA mutations, insertions and deletions and the possibility that different sequences are the same subject. The algorithm can reduce the overall size of the database to 15% of the original size. Depending on parameters, it may reduce up to 5% of the original size. Although is the same as the other platforms, the match algorithm is more sensible because it ignores the transitions and transversions, resulting in a faster way to obtain the diagnostic results. The first experiment results in an increase in speed 10 times faster than Blast while maintaining high sensitivity. This performance gain can be extended by combining other techniques already used in other studies, such as hash tables. Database URL https://github.com/ghc4/metagens.

Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of selected mutations.

The COVID-19 pandemic caused by SARS-CoV-2 has reached by February 2022 more than 380 million cases and 5.5 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the hACE2 cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in RBD have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this work, we identified 48 sites under selective pressures, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified 28 sites found not to be conditionally independent, such as E484K-N501Y. The molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for enhanced binding affinity between the spike RBD and hACE2 in P.1 and P.2 lineages (specially with L452V). Structural changes were also identified in the hACE molecule when interacting with B.1.1.7 RDB. Despite some destabilizing substitutions, a stabilizing effect was identified for the majority of the positively selected mutations.Communicated by Ramaswamy H. Sarma.

Genomic characterization and molecular evolution of SARS-CoV-2 in Rio Grande do Sul State, Brazil.

SARS-CoV-2 is the virus responsible for the COVID-19 and has afflicted the world since the end of 2019. Different lineages have been discovered and the Gamma lineage, which started the second wave of infections, was first described in Brazil, one of the most affected countries by pandemic. Therefore, this study analyzed SARS-CoV-2 sequenced genomes from Esteio city in Rio Grande do Sul, Southern Brazil. We also comparatively analyzed genomes of the two first years of the pandemic from Rio Grande do Sul state for understanding their genomic and evolutionary patterns. The phylogenomic analysis showed monophyletic groups for Alpha, Gamma, Delta and Omicron, as well as for other circulating lineages in the state. Molecular evolutionary analysis identified several sites under adaptive selection in membrane and nucleocapsid proteins which could be related to a prevalent stabilizing effect on membrane protein structure, as well as majoritarily destabilizing effects on C-terminal nucleocapsid domain.

Acute kidney injury associated with rhabdomyolysis in a patient with COVID-19.

Rhabdomyolysis is defined as the breakdown of skeletal muscle leading to the release of muscle contents into the extracellular fluid. Patients with rhabdomyolysis can be asymptomatic or have myalgia symptoms, weakness, myoglobinuria with dark urine, significant electrolyte imbalance, and acute kidney injury. Here we describe a case on acute kidney injury associated to rhabdomyolysis in a patient with COVID-19.

Comparative Genomics and Characterization of SARS-CoV-2 P.1 (Gamma) Variant of Concern From Amazonas, Brazil.

BACKGROUND: P.1 lineage (Gamma) was first described in the State of Amazonas, northern Brazil, in the end of 2020, and has emerged as a very important variant of concern (VOC) of SARS-CoV-2 worldwide. P.1 has been linked to increased infectivity, higher mortality, and immune evasion, leading to reinfections and potentially reduced efficacy of vaccines and neutralizing antibodies. METHODS: The samples of 276 patients from the State of Amazonas were sent to a central referral laboratory for sequencing by gold standard techniques, through Illumina MiSeq platform. Both global and regional phylogenetic analyses of the successfully sequenced genomes were conducted through maximum likelihood method. Multiple alignments were obtained including previously obtained unique human SARS-CoV-2 sequences. The evolutionary histories of spike and non-structural proteins from ORF1a of northern genomes were described and their molecular evolution was analyzed for detection of positive (FUBAR, FEL, and MEME) and negative (FEL and SLAC) selective pressures. To further evaluate the possible pathways of evolution leading to the emergence of P.1, we performed specific analysis for copy-choice recombination events. A global phylogenomic analysis with subsampled P.1 and B.1.1.28 genomes was applied to evaluate the relationship among samples. RESULTS: Forty-four samples from the State of Amazonas were successfully sequenced and confirmed as P.1 (Gamma) lineage. In addition to previously described P.1 characteristic mutations, we find evidence of continuous diversification of SARS-CoV-2, as rare and previously unseen P.1 mutations were detected in spike and non-structural protein from ORF1a. No evidence of recombination was found. Several sites were demonstrated to be under positive and negative selection, with various mutations identified mostly in P.1 lineage. According to the Pango assignment, phylogenomic analyses indicate all samples as belonging to the P.1 lineage. CONCLUSION: P.1 has shown continuous evolution after its emergence. The lack of clear evidence for recombination and the positive selection demonstrated for several sites suggest that this lineage emergence resulted mainly from strong evolutionary forces and progressive accumulation of a favorable signature set of mutations.

Computational studies of polyurethanases from Pseudomonas.

Polyurethanes (PU) are multifunctional polymers, used in automotive industry, in coatings, rigid and flexible foams, and also in biomimetic materials. In the same way as all plastic waste, the incorrect disposal of these materials leads to the accumulation of polyurethanes in the environment. To reduce the amount of waste as well as add value to degradation products, bioremediation methods have been studied for waste management of PU. Enzymes of the hydrolases class have been experimentally tested for enzymatic degradation of PU, with very promising results. In this work, two enzymes that can degrade polyurethanes were studied by molecular dynamics simulations: a protease and an esterase, both from Pseudomonas. From molecular dynamics simulations analysis, it was observed the stability of the structures, both in the simulations of the free enzymes and in the simulations of the complexes with a PU monomer. Hydrogen bonds were formed with the monomer and the enzymes throughout the simulation time, and the interaction free energy was found to be strongly negative, pointing to strong interactions in both cases.