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Giant viruses, with virions larger than 200 nm and genomes larger than 340 kilobase pairs, modified the now outdated perception of the virosphere. With virions now reported reaching up to 1.5 μm in size and genomes of up to 2.5 Mb encoding components shared with cellular life forms, giant viruses exhibit a complexity similar to microbes, such as bacteria and archaea. Here, we review interactions of giant viruses with their hosts and defense strategies of giant viruses against their hosts and coinfecting microorganisms or virophages. We also searched by comparative genomics for homologies with proteins described or suspected to be involved in defense mechanisms. Our search reveals that natural immunity and apoptosis seem to be crucial components of the host defense against giant virus infection. Conversely, giant viruses possess methods of hijacking host functions to counteract cellular antiviral responses. In addition, giant viruses may encode other unique and complex pathways to manipulate the host machinery and eliminate other competing microorganisms. Notably, giant viruses have evolved defense mechanisms against their virophages and they might trigger defense systems against other viruses through sequence integration. We anticipate that comparative genomics may help identifying genes involved in defense strategies of both giant viruses and their hosts. [ABSTRACT FROM AUTHOR]

Genome remodeling and exchange of sequences are widespread in the prokaryotic world and mosaic genomes challenge the classification of prokaryotes, which cannot be properly achieved in terms of a single gene or group of genes. Here, we studied individually the gene collection of the archaic microorganism Lokiarchaeum sp., suggested as an archaeal host close to the emergence of the eukaryotes. The network or rhizome of all Lokiarchaeum sp. genes revealed that the genomic repertoire is mainly composed of genes from archaeal (**36%) and bacterial origin (**28%), distantly followed by components of eukaryotic origin (**2%). Thirty-three percent of geneswere unique to this species (ORFans). Themosaicity of archaeawas also supported by studying Methanomassiliicoccus luminyensis, an archaea fromthe gut, inwhich 67%of the genomic repertoire arised fromarchaea and 22% from bacteria. Our results illustrate the intricate evolutionary relationships of the archaeal genome repertoire and highlight the rhizome-like processes of evolution in archaea, their mosaicity, and chimeric origin composed of different domains of life, questioning the reality of a tree of life. [ABSTRACT FROM AUTHOR]

Most theories on viral evolution are speculative and lack fossil comparison. Here, we isolated a modern Pithovirus-like virus from sewage samples. This giant virus, named Pithovirus massiliensis, was compared with its prehistoric counterpart, Pithovirus sibericum, found in Siberian permafrost. Our analysis revealed near-complete gene repertoire conservation, including horizontal gene transfer and ORFans. Furthermore, all orthologous genes evolved under strong purifying selection with a non-synonymous and synonymous ratio in the same range as the ratio found in the prokaryotic world. The comparison between fossil and modern Pithovirus species provided an estimation of the cadence of the molecular clock, reaching up to 3 x 10-6 mutations/site/year. In addition, the strict conservation of HGTs and ORFans in P.massiliensis revealed the stable genetic mosaicism in giant viruses and excludes the conceptof a bag of genes. The genetic stability for 30,000 years of P. massiliensis demonstrates that giant viruses evolve similarly to prokaryotes by classical mechanisms of evolution, including selection and fixation of genes, followed by selective constraints. [ABSTRACT FROM AUTHOR]

An acid-fast, rapidly growing, rod-shaped microorganism designated 8WA6 was isolated from a lake in Algiers, Algeria. The lake water was characterized by a temperature of 18 °C, a pH of 7.82, a copper concentration of 8.6 µg/L, and a cadmium concentration of 0.6 µg/L. First-line molecular identification confirmed the 8WA6 isolate to be a member of the Mycobacterium terrae complex, sharing 99.4 % 16S rRNA gene sequence similarity with M. arupense AR-30097, 98.2 % partial hsp65 gene sequence similarity with M. terrae 28K766, and 97.1 % partial rpoB gene sequence similarity with Mycobacterium sp. FI-05396. Its 4.89-Mb genome exhibits a 66.8 GC % and an average nucleotide identity of 64.5 % with M. tuberculosis, 70.5 % with M. arupense, and 75 % with M. asiaticum. In the M. terrae complex, Mycobacterium 8WA6 was unique in exhibiting growth at 42 °C, negative reaction for nitrate reduction, urease activity and Tween 80 hydrolysis, and a positive reaction for α-glucosidase and β-glucosidase. Its protein profile determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed a unique spectrum similar to M. arupense and M. terrae, exhibiting eleven specific peaks at 3787.791, 4578.019, 6349.630, 6855.638, 7202.310, 8149.608, 8775.257, 10,224.588, 10,484.116, 12,226.379, and 12,636.871 m/ z. Minimal inhibitory concentrations (MIC) for antibiotics, determined by microdilution, indicated a broad spectrum resistance, except for rifabutin (MIC, 0.5 g/L) and cefoxitin (MIC, 16 g/L). We concluded that the 8WA6 isolate is a representative isolate of a previously undescribed species in the M. terrae complex, which was named M. icosiumassiliensis sp. nov. with strain 8WA6 (Collection de Souches de l'Unité des Rickettsies, CSUR P1561, Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSM 100711) as the type strain. [ABSTRACT FROM AUTHOR]

Background Introduction of 1 Treponema pallidum complex pathogen in naive European populations following the return of Christopher Columbus' troops from Central America in 1493 is a central dogma in venereology. Methods Among skeletal elements from the seventh or eighth century uncovered in Roquevaire, France, individual RS-1003 femur macroscopically suspected of having an infectious disease was investigated by means of paleoautoimmunohistochemistry, direct metagenomics, and paleoserology, along with 1 control femur from an apparently healthy individual (R-1003) and experimental negative controls. Results RS-1003 femur showed infectious bone; paleoautoimmunohistochemistry of the lesions led to microscopic detection of a T. pallidum complex pathogen. Phylogenetic analyses comprising 71 T. pallidum complex–specific reads covering 2.37% of the T. pallidum subsp. pallidum reference genome sequence revealed an ancestral T. pallidum complex pathogen in the lesion. Paleoserology detecting T. pallidum –specific antigens confirmed positive serological findings in individual RS-1003. Individual R-1003 and the negative controls remained negative. Conclusions This case, predating by 8 centuries previous detections of T. pallidum complex treponematosis in Europe, indicated that European populations were not naive to these pathogens before the 1493 introduction of a Central American T. pallidum complex pathogen overwhelming the T. pallidum ones previously circulating in the Old World. These data break a century-old dogma in medical microbiology. [ABSTRACT FROM AUTHOR]

Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus ( PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L, which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M s toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical intermediates was clearly demonstrated, which raises prospects for applying this enzyme to detoxify toxic compounds formed during the degradation of lignin. [ABSTRACT FROM AUTHOR]

Mycoviruses are viruses that selectively infect and multiply in fungal cells. Malassezia is the most abundant fungus on human skin and is associated with a variety of conditions, including atopic eczema, atopic dermatitis, dandruff, folliculitis, pityriasis versicolor, and seborrheic dermatitis. Here, we conducted mycovirome studies on 194 public transcriptomes of Malassezia (2,568,212,042 paired-end reads) screened against all available viral proteins. Transcriptomic data were assembled de novo resulting in 1,170,715 contigs and 2,995,306 open reading frames (ORFs) that were subsequently tracked for potential viral sequences. Eighty-eight virus-associated ORFs were detected in 68 contigs from 28 Sequence Read Archive (SRA) samples. Seventy-five and thirteen ORFs were retrieved from transcriptomes of Malassezia globosa and Malassezia restricta, respectively. Phylogenetic reconstructions revealed three new mycoviruses belonging to the Totivirus genus and named Malassezia globosa-associated-totivirus 1 (MgaTV1); Malassezia restricta-associated-totivirus 1 (MraTV1) and Malassezia restricta-associated-totivirus 2 (MraTV2). These viral candidates extend our understanding of the diversity and taxonomy of mycoviruses as well as their co-evolution with their fungal hosts. These results reflected the unexpected diversity of mycoviruses hidden in public databases. In conclusion, this study sheds light on the discovery of novel mycoviruses and opens the door to study their impact on disease caused by the host fungus Malassezia and globally, their implication in clinical skin disorders. [ABSTRACT FROM AUTHOR]

Background Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. Results The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. Indepth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. Conclusions With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Fungi compete against each other for environmental resources. These interspecific combative interactions encompass a wide range of mechanisms. In this study, we highlight the ability of the white-rot fungus Pycnoporus coccineus to quickly overgrow or replace a wide range of competitor fungi, including the gray-mold fungus Botrytis cinerea and the brown-rot fungus Coniophora puteana. To gain a better understanding of the mechanisms deployed by P. coccineus to compete against other fungi and to assess whether common pathways are used to interact with different competitors, differential gene expression in P. coccineus during cocultivation was assessed by transcriptome sequencing and confirmed by quantitative reverse transcription-PCR analysis of a set of 15 representative genes. Compared with the pure culture, 1,343 transcripts were differentially expressed in the interaction with C. puteana and 4,253 were differentially expressed in the interaction with B. cinerea, but only 197 transcripts were overexpressed in both interactions. Overall, the results suggest that a broad array of functions is necessary for P. coccineus to replace its competitors and that different responses are elicited by the two competitors, although a portion of the mechanism is common to both. However, the functions elicited by the expression of specific transcripts appear to converge toward a limited set of roles, including detoxification of secondary metabolites. [ABSTRACT FROM AUTHOR]

Background: Since its inception, the carbohydrate-active enzymes database (CAZy; www.cazy.org) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended noncatalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category. Results: Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name "Auxiliary Activities" in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot). Conclusions: The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery. [ABSTRACT FROM AUTHOR]

The chordate proteome history database (http://ioda.univ-provence.fr) comprises some 20,000 evolutionary analyses of proteins from chordate species. Our main objective was to characterize and study the evolutionary histories of the chordate proteome, and in particular to detect genomic events and automatic functional searches. Firstly, phylogenetic analyses based on high quality multiple sequence alignments and a robust phylogenetic pipeline were performed for the whole protein and for each individual domain. Novel approaches were developed to identify orthologs/paralogs, and predict gene duplication/gain/loss events and the occurrence of new protein architectures (domain gains, losses and shuffling). These important genetic events were localized on the phylogenetic trees and on the genomic sequence. Secondly, the phylogenetic trees were enhanced by the creation of phylogroups, whereby groups of orthologous sequences created using OrthoMCL were corrected based on the phylogenetic trees; gene family size and gene gain/loss in a given lineage could be deduced from the phylogroups. For each ortholog group obtained from the phylogenetic or the phylogroup analysis, functional information and expression data can be retrieved. Database searches can be performed easily using biological objects: protein identifier, keyword or domain, but can also be based on events, eg, domain exchange events can be retrieved. To our knowledge, this is the first database that links group clustering, phylogeny and automatic functional searches along with the detection of important events occurring during genome evolution, such as the appearance of a new domain architecture. [ABSTRACT FROM AUTHOR]

Understanding the evolutionary plasticity of the genome requires a global, comparative approach in which genetic events are considered both in a phylogenetic framework and with regard to population genetics and environmental variables. In the mechanisms that generate adaptive and non-adaptive changes in genomes, segmental duplications (duplication of individual genes or genomic regions) and polyploidization (whole genome duplications) are well-known driving forces. The probability of fixation and maintenance of duplicates depends on many variables, including population sizes and selection regimes experienced by the corresponding genes: a combination of stochastic and adaptive mechanisms has shaped all genomes. A survey of experimental work shows that the distinction made between fixation and maintenance of duplicates still needs to be conceptualized and mathematically modeled. Here we review the mechanisms that increase or decrease the probability of fixation or maintenance of duplicated genes, and examine the outcome of these events on the adaptation of the organisms. [ABSTRACT FROM AUTHOR]

Abstract: The fhbA genes encoding putative flavohemoglobins (FHb) from Aspergillus niger and Aspergillus oryzae were isolated. Comparison of the deduced amino acid sequence of the A. niger fhbA gene and other putative filamentous fungal FHb-encoding genes to that of Ralstonia eutropha shows an overall conserved gene structure and completely conserved catalytic amino acids. Several yeasts and filamentous fungi, including both Aspergillus species have been found to contain a small FHb gene family mostly consisting of two family members. Based on these sequences the evolutionary history of the fungal FHb family was reconstructed. The isolated fhbA genes from A. oryzae and A. niger belong to a phylogenetic group, which exclusively contains Aspergillus genes. Different experimental approaches show that fhbA transcript levels appear during active hyphal growth. Moreover, in a pclA-disrupted strain with a hyperbranching growth phenotype, the transcript levels of the fhbA gene were 2–5 times higher compared to the wild-type. These results suggest that FHb from filamentous fungi have a function that is correlated to the hyphal growth phenotype. [Copyright &y& Elsevier]

Abstract: The breakdown of lignin by fungi is a key step during carbon recycling in terrestrial ecosystems. This process is of great interest for green and white biotechnological applications. Given the importance of these enzymatic processes, we have classified the enzymes potentially involved in lignin catabolism into sequence-based families and integrated them in a newly developed database, designated Fungal Oxidative Lignin enzymes (FOLy). Families were defined after sequence similarity searches starting from protein sequences and validated by the convergence of results with biochemical experiments reported in the literature. The resulting database was applied as a tool for the functional annotation of genomes from different fungi, namely (i) the Basidiomycota Coprinopsis cinerea, Phanerochaete chrysosporium and Ustilago maydis and (ii) the Ascomycota Aspergillus nidulans and Trichoderma reesei. Genomic comparison of the oxidoreductases of these fungi revealed significant differences in the putative enzyme arsenals. Two Ascomycota fungal genomes were annotated and new candidate genes were identified that could be useful for lignin degradation and (or) melanin synthesis, and their function investigated experimentally. This database efforts aims at providing the means to get new insights for the understanding and biotechnological exploitation of the lignin degradation. A WWW server giving access to the routinely updated FOLy classifications of enzymes potentially involved in lignin degradation can be found at http://foly.esil.univ-mrs.fr. [Copyright &y& Elsevier]

Background: The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily. Results: Using an in silico approach, we demonstrated that GPR50 is the ortholog of the high affinity Mel1c receptor. It was necessary to also study the synteny of this gene to reach this conclusion because classical mathematical models that estimate orthology and build phylogenetic trees were not sufficient. The receptor has been deeply remodelled through evolution by the mutation of numerous amino acids and by the addition of a long C-terminal tail. These alterations have modified its affinity for melatonin and probably affected its interactions with the other two known melatonin receptors MT1 and MT2 that are encoded by Mel1a and Mel1b genes respectively. Evolutionary studies provided evidence that the GPR50 group evolved under different selective pressure as compared to the orthologous groups Me11 a, b, and c. Conclusion: This study demonstrated that there are only three members in the melatonin receptor subfamily with one of them (Me11c) undergoing rapid evolution from fishes and birds to mammals. Further studies are necessary to investigate the physiological roles of this receptor. [ABSTRACT FROM AUTHOR]

The recent availability of the complete genome sequences of a large number of model organisms, together with the immense amount of data being produced by the new high-throughput technologies, means that we can now begin comparative analyses to understand the mechanisms involved in the evolution of the genome and their consequences in the study of biological systems. Phylogenetic approaches provide a unique conceptual framework for performing comparative analyses of all this data, for propagating information between different systems and for predicting or inferring new knowledge. As a result, phylogeny-based inference systems are now playing an increasingly important role in most areas of high throughput genomics, including studies of promoters (phylogenetic footprinting), interactomes (based on the presence and degree of conservation of interacting proteins), and in comparisons of transcriptomes or proteomes (phylogenetic proximity and co-regulation/co-expression). Here we review the recent developments aimed at making automatic, reliable phylogenybased inference feasible in large-scale projects. We also discuss how evolutionary concepts and phylogeny-based inference strategies are now being exploited in order to understand the evolution and function of biological systems. Such advances will be fundamental for the success of the emerging disciplines of systems biology and synthetic biology, and will have wide-reaching effects in applied fields such as biotechnology, medicine and pharmacology. [ABSTRACT FROM AUTHOR]

To demonstrate that a given change in the environment has contributed to the emergence of a given genotypic and phenotypic shift during the course of evolution, one should ask to what extent such shifts would have occurred without environmental change. Of course, such tests are rarely practical but phenotypic novelties can still be correlated to genomic shifts in response to environmental changes if enough information is available. We surveyed and re-evaluated the published data in order to estimate the role of environmental changes on the course of species and genomic evolution. Only a few published examples clearly demonstrate a causal link between a given environmental change and the fixation of a genomic variant resulting in functional modification (gain, loss or alteration of function). Many others suggested a link between a given phenotypic shift and a given environmental change but failed to identify the underlying genomic determinant(s) and/or the associated functional consequence(s). The proportion of genotypic and phenotypic variation that is fixed concomitantly with environmental changes is often considered adaptive and hence, the result of positive selection, even though alternative causes, such as genetic drift, are rarely investigated. Therefore, the second aim herein is to review evidence for the mechanisms leading to fixation. [ABSTRACT FROM AUTHOR]

Abstract: By using in silico methods in a previous study, we identified 100 oocyte-specific genes and 150 genes, enriched in the mouse oocyte. Interestingly, approximately half of the oocyte-specific genes tend to cluster on mouse chromosomes as if they have recently duplicated during evolution. In this study, we focused our attention on mouse BRDT, which belongs to a family of four structurally related proteins characterized by two N-terminal bromodomains and one C-terminal extraterminal domain (ET domain), defining the BET family. In mammals, BRD2, -3, and -4 are ubiquitously expressed, whereas BRDT expression was shown to be restricted to the testis. We were interested to know whether there was a correlation between the evolutionary rate and the specificity of expression of these four paralogous genes. First, we show by RT-PCR and in situ hybridization that BRDT is also expressed in mouse oocyte. Moreover, phylogenetic analyses show that the BRDT germ cell-specific orthology group clearly evolves faster than its ubiquitously expressed paralogs BRD2, BRD3, and BRD4. This suggests that there is a relationship between the evolution of these four groups of orthology and their tissue specificity of expression. [Copyright &y& Elsevier]

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l−1 in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest. [ABSTRACT FROM AUTHOR]

Background: There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/feruloyl esterase A family, whose functional diversification makes it a very promising candidate. Results: The results suggested functional shift following a duplication event where neofunctionalisation of feruloyl esterase A had occurred with conservation of the ancestral lipase function. Evolutionary shift was detected using the branch-site model for testing positive selection on individual codons along specific lineages. Positively selected amino acids were detected. Furthermore, biological data obtained from site-directed mutagenesis experiments clearly demonstrated that certain amino acids under positive selection were involved in the functional shift. We reassessed evolutionary history in terms of environmental response, and hypothesized that environmental changes such as colonisation by terrestrial plants might have driven adaptation by functional diversification in Euascomycetes (Aspergilli), thus conferring a selective advantage on this group. Conclusion: The results reported here illustrate a rare example of connection between fundamental events in molecular evolution. We demonstrated an unequivocal connection between evolutionary and functional shifts, which led us to conclude that these events were probably linked to environmental change. [ABSTRACT FROM AUTHOR]

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAFA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immuno-detection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physico-chemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a Kd of 9.9 × 10-8 M for the dissociation constant and 0.98 μmol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates. [ABSTRACT FROM AUTHOR]

The xynB gene, which encodes endo-β-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg·L–1. The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 °C with stability in the pH range 4.0-7.0 and temperature up to 50 °C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg·mL–1 and 3881 U·mg–1, respectively. [ABSTRACT FROM AUTHOR]

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin. [ABSTRACT FROM AUTHOR]

The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease deficient A. niger strain. Homologous overproduction allows to reach an esterase activity of 18 nkat mL-1 against MCA as substrate. The improvement factor was 16-fold higher as compared to the production level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was estimated to be around 100 mg L-1. Recombinant FAEB was purified 14.6-fold to homogeneity from an 8-day-old culture by a single affinity chromatographic step with a recovery of 64%. SDS–PAGE revealed a single band with a molecular mass of 75 kDa, while under non-denatured conditions, native enzyme has a molecular mass of around 150 kDa confirming that the recombinant FAEB is a homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 °C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 °C for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caffeoyl quinic acid) were as follows: Km: 0.13, 0.029, and 0.16 mM and Vmax: 1101, 527.6, and 28.3 nkat mg-1, respectively. This is the first report on the homologous overproduction of feruloyl esterase B in A. niger. [Copyright &y& Elsevier]

Surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic epidemiology led us to detect several variants since summer 2020. We report the recent spread of a new SARS-CoV-2 spike 501Y variant. SARS-CoV-2 sequences obtained from human nasopharyngeal samples by Illumina next-generation sequencing were analysed using Nextclade and an in-house Python script and were compared using BLASTn to the GISAID database. Phylogeny was investigated using the IQ-TREE software. We identified that SARS-CoV-2 genomes from four patients diagnosed in our institute harboured a new set of amino acid substitutions including L18F, L452R, N501Y, A653V, H655Y, D796Y, G1219V ± Q677H. These spike N501Y genomes are the first of Nextstrain clade 19B. We obtained partial spike gene sequences of this genotype for an additional 43 patients. All patients infected with this genotype were diagnosed since mid-January 2021. We detected 42 other genomes of this genotype in GISAID, which were obtained from samples collected in December 2020 in four individuals and in 2021 in 38 individuals. The 89 sequences obtained in our institute or other laboratories originated from the Comoros archipelago, western European countries (mostly metropolitan France), Turkey and Nigeria. These findings warrant further studies to investigate the spread, epidemiological and clinical features, and sensitivity to immune responses of this variant. [ABSTRACT FROM AUTHOR]

The symbiotic Wolbachia are the most sophisticated mutualistic bacterium among all insect-associated microbiota. Wolbachia-insect relationship fluctuates from the simple facultative/parasitic to an obligate nutritional-mutualistic association as it was the case of the bedbug-Wolbachia from Cimexlectularius. Understanding this association may help in the control of associated arthropods. Genomic data have proven to be reliable tools in resolving some aspects of these symbiotic associations. Although, Wolbachia appear to be fastidious or uncultivated bacteria which strongly limited their study. Here we proposed Drosophila S2 cell line for the isolation and culture model to study Wolbachia strains. We therefore isolated and characterized a novel Wolbachia strain associated with the bedbug Cimexhemipterus, designated as wChem strain PL13, and proposed Wolbachiamassiliensis sp. nov. strain wChem-PL13 a type strain of this new species from new supergroup T. Phylogenetically, T-supergroup was close to F and S-supergroups from insects and D-supergroup from filarial nematodes. We determined the 1,291,339-bp genome of wChem-PL13, which was the smallest insect-associated Wolbachia genomes. Overall, the wChem genome shared 50% of protein coding genes with the other insect-associated facultative Wolbachia strains. These findings highlight the diversity of Wolbachia genotypes as well as the Wolbachia-host relationship among Cimicinae subfamily. The wChem provides folate and riboflavin vitamins on which the host depends, while the bacteria had a limited translation mechanism suggesting its strong dependence to its hosts. However, the clear-cut distinction between mutualism and parasitism of the wChem in C. hemipterus cannot be yet ruled out. [ABSTRACT FROM AUTHOR]

In an epidemiological study, mimivirus was reported as an unlikely cause of human respiratory infections in China. Our analysis revealed the nonsensitivity of the PCR method, which detected less than 10% of the current known mimivirus. We conclude that epidemiologic studies must use accurate and sensitive laboratory test methods. [ABSTRACT FROM AUTHOR]

• The severe acute respiratory syndrome (SARS-CoV-2) Marseille-4 variant caused an epidemic that started in August and is still ongoing. • This variant harbours 13 hallmark mutations, including one in the spike receptor binding domain. • The variant predominated in Marseille from September 2020 and caused a re-infection in 11 patients. • Hypoxemia was more frequent than with clade 20A strains that circulated before May 2020. • The sudden appearance of Marseille-4 points towards an animal reservoir, possibly mink. In Marseille, France, following a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in March–May 2020, a second epidemic phase occurred from June, involving 10 new variants. The Marseille-4 variant caused an epidemic that started in August and is still ongoing. The 1038 SARS-CoV-2 whole genome sequences obtained in our laboratory by next-generation sequencing with Illumina technology were analysed using Nextclade and nextstrain/ncov pipelines and IQ-TREE. A Marseille-4-specific qPCR assay was implemented. Demographic and clinical features were compared between patients with the Marseille-4 variant and those with earlier strains. Marseille-4 harbours 13 hallmark mutations. One leads to an S477N substitution in the receptor binding domain of the spike protein targeted by current vaccines. Using a specific qPCR, it was observed that Marseille-4 caused 12–100% of SARS-CoV-2 infections in Marseille from September 2020, being involved in 2106 diagnoses. This variant was more frequently associated with hypoxemia than were clade 20A strains before May 2020. It caused a re-infection in 11 patients diagnosed with different SARS-CoV-2 strains before June 2020, suggesting either short-term protective immunity or a lack of cross-immunity. Marseille-4 should be considered as a major SARS-CoV-2 variant. Its sudden appearance points towards an animal reservoir, possibly mink. The protective role of past exposure and current vaccines against this variant should be evaluated. [ABSTRACT FROM AUTHOR]

Presence of Leishmania spp. was evaluated in the blood of nine red howler monkeys (Alouatta seniculus) from a specific area of French Guiana, located in the northeast of the Amazon. The molecular detection was performed based on PCR targeting the markers 18S rRNA, kDNA and ITS2 genes, as well as rapid immunomigration tests. Two monkeys were positive for Leishmania infantum and one for Leishmania guyanensis. While L. guyanensis cutaneous leishmaniasis is common, visceral leishmaniasis (human and canine) caused by L. infantum has never been described in this area. The howler monkey proved to be a sentinel and a potential reservoir of a serious zoonosis. These results must be carefully considered by public health officials and veterinarians in the future. [ABSTRACT FROM AUTHOR]

β-lactamases are enzymes which are commonly produced by bacteria and which degrade the β-lactam ring of β-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of β-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of b-lactamases (A, B, C, and D), which were used to identify potential β-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI's nonredundant databases (44101). Our analysis revealed the existence of putative β-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A-D). This is the first report on the large-scale phylogenetic diversity of new members of β-lactamases, and our results revealed that metagenomic database dark-matter contains β-lactamase-like antibiotic resistance genes. [ABSTRACT FROM AUTHOR]

Microbial culturomics is a new subfield of postgenomic medicine and omics biotechnology application that has broadened our awareness on bacterial diversity of the human microbiome, including the human vaginal flora bacterial diversity. Using culturomics, a new obligate anaerobic Gram-stain-negative rod-shaped bacterium designated strain khD1T was isolated in the vagina of a patient with bacterial vaginosis and characterized using taxonogenomics. The most abundant cellular fatty acids were C15:0 anteiso (36%), C16:0 (19%), and C15:0 iso (10%). Based on an analysis of the full-length 16S rRNA gene sequences, phylogenetic analysis showed that the strain khD1T exhibited 90% sequence similarity with Prevotella loescheii, the phylogenetically closest validated Prevotella species. With 3,763,057 bp length, the genome of strain khD1T contained (mol%) 48.7 G+ C and 3248 predicted genes, including 3194 protein-coding and 54 RNA genes. Given the phenotypical and biochemical characteristic results as well as genome sequencing, strain khD1T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella lascolaii sp. nov. is proposed. The type strain is khD1T ( = CSUR P0109, =DSM 101754). These results show that microbial culturomics greatly improves the characterization of the human microbiome repertoire by isolating potential putative new species. Further studies will certainly clarify the microbial mechanisms of pathogenesis of these new microbes and their role in health and disease. Microbial culturomics is an important new addition to the diagnostic medicine toolbox and warrants attention in future medical, global health, and integrative biology postgraduate teaching curricula. [ABSTRACT FROM AUTHOR]

Having previously shown that soluble E-cadherin (sE-cad) is found in sera of Q fever patients and that infection of BeWo cells by C. burnetii leads to modulation of the E-cad/β-cat pathway, our purpose was to identify which sheddase(s) might catalyze the cleavage of E-cad. Here, we searched for a direct mechanism of cleavage initiated by the bacterium itself, assuming the possible synthesis of a sheddase encoded in the genome of C. burnetii or an indirect mechanism based on the activation of a human sheddase. Using a straightforward bioinformatics approach to scan the complete genomes of four laboratory strains of C. burnetii, we demonstrate that C. burnetii encodes a 451 amino acid sheddase (CbHtrA) belonging to the HtrA family that is differently expressed according to the bacterial virulence. An artificial CbHtrA gene (CoxbHtrA) was expressed, and the CoxbHtrA recombinant protein was found to have sheddase activity. We also found evidence that the C. burnetii infection triggers an over-induction of the human HuHtrA gene expression. Finally, we demonstrate that cleavage of E-cad by CoxbHtrA on macrophages-THP-1 cells leads to an M2 polarization of the target cells and the induction of their secretion of IL-10, which "disarms" the target cells and improves C. burnetii replication. Taken together, these results demonstrate that the genome of C. burnetii encodes a functional HtrA sheddase and establishes a link between the HtrA sheddase-induced cleavage of E-cad, the M2 polarization of the target cells and their secretion of IL-10, and the intracellular replication of C. burnetii. [ABSTRACT FROM AUTHOR]

Chemical decomposition and fragmentation may limit the detection of ancient host and microbial DNA while some proteins can be detected for extended periods of time. We applied paleoproteomics on 300-year-old dental pulp specimens recovered from 16 individuals in two archeological funeral sites in France, comprising one documented plague site and one documented plague-negative site. The dental pulp paleoproteome of the 16 teeth comprised 439 peptides representative of 30 proteins of human origin and 211 peptides representative of 27 proteins of non-human origin. Human proteins consisted of conjunctive tissue and blood proteins including IgA immunoglobulins. Four peptides were indicative of three presumable Yersinia pestis proteins detected in 3/8 dental pulp specimens from the plague-positive site but not in the eight dental pulp specimens collected in the plague-negative site. Paleoproteomics applied to the dental pulp is a new and innovative approach to screen ancient individuals for the detection of blood-borne pathogens and host inflammatory response. [ABSTRACT FROM AUTHOR]

Background Louse-borne trench fever caused by Bartonella quintana is a neglected public health concern, known to be transmitted from body louse feces via scratching. No viable B. quintana have ever been isolated from head lice before; therefore, their role as a vector is still poorly understood. Methods In Senegal, the implementation of a permanent local surveillance system in a point-of-care laboratory (POC) allows the monitoring of emerging diseases. Here we used culture as well as molecular and genomic approaches to document an outbreak of trench fever associated with head lice in the village of Ndiop. Head lice and blood samples were collected from febrile patients between November 2010 and April 2015. Genomes of 2 isolated strains of B. quintana were sequenced and analyzed. Results A total of 2289 blood samples were collected in the 2010–2015 period. From 2010–2013, B. quintana DNA was detected by polymerase chain reaction (PCR) in 0.25% (4/1580). In 2014, 228 blood samples were collected, along with 161 head lice from 5 individuals. B. quintana DNA was detected in 4.4% (10/228) of blood samples, and in lice specimens collected from febrile patients (61.7%, 50/81) and non-febrile patients (61.4%, 43/70). Two B. quintana strains were isolated from blood and head lice from 2 different patients. Genomic sequence analysis showed 99.98% overall similarity between both strains. Conclusions The presence of live B. quintana in head lice, and the genetic identity of strains from patients' blood and head lice during a localized outbreak in Senegal, supports the evidence of head lice vectorial capacity. [ABSTRACT FROM AUTHOR]

Infection by Coxiella burnetii, the etiological agent of Q fever, poses the risk of causing severe obstetrical complications in pregnant women. C. burnetii is known for its placental tropism based on animal models of infection. The Nine Mile strain has been mostly used to study C. burnetii pathogenicity but the contribution of human isolates to C. burnetii pathogenicity is poorly understood. In this study, we compared five C. burnetii isolates from human placentas with C. burnetii strains including Nine Mile (NM) as reference. Comparative genomic analysis revealed that the Cb122 isolate was distinct from other placental isolates and the C. burnetii NM strain with a set of unique genes involved in energy generation and a type 1 secretion system. The infection of Balb/C mice with the Cb122 isolate showed higher virulence than that of NM or other placental isolates. We evaluated the pathogenicity of the Cb122 isolate by in vitro and ex vivo experiments. As C. burnetii is known to infect and survive within macrophages, we isolated monocytes and placental macrophages from healthy donors and infected them with the Cb122 isolate and the reference strain. We showed that bacteria from the Cb122 isolate were less internalized by monocyte-derived macrophages (MDM) than NM bacteria but the reference strain and the Cb122 isolate were similarly internalized by placental macrophages. The Cb122 isolate and the reference strain survived similarly in the two macrophage types. While the Cb122 isolate and the NM strain stimulated a poorly inflammatory program in MDM, they elicited an inflammatory program in placenta macrophages. We also reported that the Cb122 isolate and NM strain were internalized by trophoblastic cell lines and primary trophoblasts without specific replicative profiles. Placental explants were then infected with the Cb122 isolate and the NM strain. The bacteria from the Cb122 isolate were enriched in the chorionic villous foetal side. It is likely that the Cb122 isolate exhibited increased virulence in the multicellular environment provided by explants. Taken together, these results showed that the placental isolate of C. burnetii exhibits a specific infectious profile but its pathogenic role is not as high as the host immune response in pregnant women. [ABSTRACT FROM AUTHOR]

Giant viruses infect protozoa, especially amoebae of the genus Acanthamoeba . These viruses possess genetic elements named Mobilome. So far, this mobilome comprises provirophages which are integrated into the genome of their hosts, transpovirons, and Maverick/Polintons. Virophages replicate inside virus factories within Acanthamoeba and can decrease the infectivity of giant viruses. The virophage infecting CroV was found to be integrated in the host of CroV, Cafeteria roenbergensis , thus protecting C. roenbergensis by reduction of CroV multiplication. Because of this unique property, assessment of the mechanisms of replication of virophages and their relationship with giant viruses is a key element of this investigation. This work aimed at evaluating the presence and the dynamic of these mobile elements in sixteen Acanthamoeba genomes. No significant traces of the integration of genomes or sequences from known virophages were identified in all the available Acanthamoeba genomes. These results brought us to hypothesize that the interactions between mimiviruses and their virophages might occur through different mechanisms, or at low frequency. An additional explanation could be that our knowledge of the diversity of virophages is still very limited. [ABSTRACT FROM AUTHOR]

At the time of a new and unprecedented viral pandemic, many questions are being asked about the genomic evolution of SARS-CoV-2 and the emergence of different variants, leading to therapeutic and immune evasion and survival of this genetically highly labile RNA virus. The nasopharyngeal persistence of infectious virus beyond 17 days proves its constant interaction with the human immune system and increases the intra-individual mutational possibilities. We performed a prospective high-throughput sequencing study (ARTIC Nanopore) of SARS-CoV-2 from so-called "persistent" patients, comparing them with a non-persistent population, and analyzing the quasi-species present in a single sample at time t. Global intra-individual variability in persistent patients was found to be higher than in controls (mean 5.3%, Standard deviation 0.9 versus 4.6% SD 0.3, respectively, p < 0.001). In the detailed analysis, we found a greater difference between persistent and non-persistent patients with non-severe COVID 19, and between the two groups infected with clade 20A. Furthermore, we found minority N501Y and P681H mutation clouds in all patients, with no significant differences found both groups. The question of the SARS-CoV-2 viral variants' genesis remains to be further investigated, with the need to prevent new viral propagations and their consequences, and quasi-species analysis could be an important key to watch out. [ABSTRACT FROM AUTHOR]

Since the start of the coronavirus disease of 2019 (COVID‐19) pandemic, several episodes of human‐to‐animal severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) transmission have been described in different countries. The role of pets, especially domestic dogs, in the COVID‐19 epidemiology is highly questionable and needs further investigation. In this study, we report a case of COVID‐19 in a French dog living in close contact with its owners who were COVID‐19 patients. The dog presented rhinitis and was sampled 1 week after its owners (a man and a woman) were tested positive for COVID‐19. The nasal swabs for the dog tested remained positive for SARS‐CoV‐2 by reverse transcription quantitative real‐time PCR (RT‐qPCR) 1 month following the first diagnosis. Specific anti‐SARS‐CoV‐2 antibodies were detectable 12 days after the first diagnosis and persisted for at least 5 months as tested using enzyme‐linked immunoassay (ELISA) and automated western blotting. The whole‐genome sequences from the dog and its owners were 99%–100% identical (with the man and the woman's sequences, respectively) and matched the B.1.160 variant of concern (Marseille‐4 variant), the most widespread in France at the time the dog was infected. This study documents the first detection of B.1.160 in pets (a dog) in France, and the first canine genome recovery of the B.1.160 variant of global concern. Moreover, given the enhanced infectivity and transmissibility of the Marseille‐4 variant for humans, this case also highlights the risk that pets may potentially play a significant role in SARS‐CoV‐2 outbreaks and may transmit the infection to humans. We have evidence of human‐to‐dog transmission of the Marseille‐4 variant since the owners were first to be infected. Finally, owners and veterinarians must be vigilent for canine COVID‐19 when dogs are presented with respiratory clinical signs. [ABSTRACT FROM AUTHOR]

Genetic recombination is a major evolutionary mechanism among RNA viruses, and it is common in coronaviruses, including those infecting humans. A few SARS-CoV-2 recombinants have been reported to date whose genome harbored combinations of mutations from different mutants or variants, but only a single patient's sample was analyzed, and the virus was not isolated. Here, we report the gradual emergence of a hybrid genome of B.1.160 and Alpha variants in a lymphoma patient chronically infected for 14 months, and we isolated the recombinant virus. The hybrid genome was obtained by next-generation sequencing, and the recombination sites were confirmed by PCR. This consisted of a parental B.1.160 backbone interspersed with two fragments, including the spike gene, from an Alpha variant. An analysis of seven sequential samples from the patient decoded the recombination steps, including the initial infection with a B.1.160 variant, then a concurrent infection with this variant and an Alpha variant, the generation of hybrid genomes, and eventually the emergence of a predominant recombinant virus isolated at the end of the patient's follow-up. This case exemplifies the recombination process of SARS-CoV-2 in real life, and it calls for intensifying the genomic surveillance in patients coinfected with different SARS-CoV-2 variants, and more generally with several RNA viruses, as this may lead to the appearance of new viruses. [ABSTRACT FROM AUTHOR]

Over the past two years, several variants of SARS-CoV-2 have emerged and spread all over the world. However, infectivity, clinical severity, re-infection, virulence, transmissibility, vaccine responses and escape, and epidemiological aspects have differed between SARS-CoV-2 variants. Currently, very few treatments are recommended against SARS-CoV-2. Identification of effective drugs among repurposing FDA-approved drugs is a rapid, efficient and low-cost strategy against SARS-CoV-2. One of those drugs is ivermectin. Ivermectin is an antihelminthic agent that previously showed in vitro effects against a SARS-CoV-2 isolate (Australia/VI01/2020 isolate) with an IC50 of around 2 µM. We evaluated the in vitro activity of ivermectin on Vero E6 cells infected with 30 clinically isolated SARS-CoV-2 strains belonging to 14 different variants, and particularly 17 strains belonging to six variants of concern (VOC) (variants related to Wuhan, alpha, beta, gamma, delta and omicron). The in vitro activity of ivermectin was compared to those of chloroquine and remdesivir. Unlike chloroquine (EC50 from 4.3 ± 2.5 to 29.3 ± 5.2 µM) or remdesivir (EC50 from 0.4 ± 0.3 to 25.2 ± 9.4 µM), ivermectin showed a relatively homogeneous in vitro activity against SARS-CoV-2 regardless of the strains or variants (EC50 from 5.1 ± 0.5 to 6.7 ± 0.4 µM), except for one omicron strain (EC50 = 1.3 ± 0.5 µM). Ivermectin (No. EC50 = 219, mean EC50 = 5.7 ± 1.0 µM) was, overall, more potent in vitro than chloroquine (No. EC50 = 214, mean EC50 = 16.1 ± 9.0 µM) (p = 1.3 × 10−34) and remdesivir (No. EC50 = 201, mean EC50 = 11.9 ± 10.0 µM) (p = 1.6 × 10−13). These results should be interpreted with caution regarding the potential use of ivermectin in SARS-CoV-2-infected patients: it is difficult to translate in vitro study results into actual clinical treatment in patients. [ABSTRACT FROM AUTHOR]

Reconstructing the evolutionary history of all species is an essential objective for evolutionary biologists. Much effort has been devoted to ancestral genome reconstruction. Numbered genome sequencing of current and extinct organisms enables evolutionary biologists to compare genomic data and reconstruct ancestral genomes. Long-term conservation of karyotype, gene order or gene sequence are clues to the heritage of each species and these data can be used by evolutionary biologists to synthesize distant ancestral genomes. In this review, we referred to the recent advances in ancestral genomes reconstruction and the insight it gives on genome evolution. Special attention is devoted to the use of this knowledge to understand the evolution of the immune system genes. [Copyright &y& Elsevier]

We detected SARS-CoV-2 of PANGO lineage R.1 with the spike substitution E484K in three patients. Eleven other sequences in France and 8,831 worldwide were available from GISAID, 92% originating from Japan. The three genome sequences from our institute were phylogenetically closest to another from Guinea-Conakry, where one of the patients had travelled. These viruses did not exhibit any unusual features in cell culture. Spike structural predictions indicated a 1.3-time higher transmissibility index than for the globally spread B.1.1.7 variant but also an affinity loss for gangliosides that might have slowed dissemination. The spread of new SARS-CoV-2 mutants/variants is still not well understood and therefore difficult to predict, and this hinders implementation of effective preventive measures, including adapted vaccines. [ABSTRACT FROM AUTHOR]

Strain Marseille-P4119T was isolated from a faecal sample of a healthy 32-year-old faecal transplant donor. The bacterium was anaerobic, Gram-negative, rod-shaped, non-motile, and did not produce spores. We studied its phenotypic characteristics and sequenced its whole genome. The major fatty acids were C15:0anteiso and C15:0iso. The final genome assembly was 3912650 bp long with a 44.4 mol% G + C content, 3094 protein-coding genes and 74 RNA genes. Strain Marseille-P4119T exhibited a 97.10% 16S rRNA sequence identity and a 29.0% dDDH with Prevotella stercorea CB35T, OrthoANI values ranged from 68.5% with Prevotella enoeca to 77.4% with Prevotella stercorea , the phylogenetically closest bacterial species with standing in nomenclature. Based on the phylogenetic, phenotypic and genomic analyses, we propose the creation of the novel species Prevotella merdae sp. nov. The type strain is Marseille-P4119T (= CSUR P4119T = CECT 9566T). [ABSTRACT FROM AUTHOR]

To compare the demographics, clinical characteristics and severity of patients infected with nine different SARS-CoV-2 variants, during three phases of the COVID-19 epidemic in Marseille. A single centre retrospective cohort study was conducted in 1760 patients infected with SARS-CoV-2 of Nextstrain clades 20A, 20B, and 20C (first phase, February–May 2020), Pangolin lineages B.1.177 (we named Marseille-2) and B.1.160 (Marseille-4) variants (second phase, June–December 2020), and B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and A.27 (Marseille-501) variants (third phase, January 2021-today). Outcomes were the occurrence of clinical failures, including hospitalisation, transfer to the intensive-care unit, and death. During each phase, no major differences were observed with regards to age and gender distribution, the prevalence of chronic diseases, and clinical symptoms between variants circulating in a given phase. The B.1.177 and B.1.160 variants were associated with more severe outcomes. Infections occurring during the second phase were associated with a higher rate of death as compared to infections during the first and third phases. Patients in the second phase were more likely to be hospitalised than those in the third phase. Patients infected during the third phase were more frequently obese than others. A large cohort study is recommended to evaluate the transmissibility and to better characterise the clinical severity of emerging variants. • Monitoring the epidemiology of SARS-CoV-2 variants is very important for epidemic control • No major differences were observed between COVID-19 patients infected with variants circulating in each phase of epidemic. • Infections occurring in the second phase were associated with a higher mortality compared with the first and third episodes • More research on clinical severity of COVID-19 in patients infected with variants harbouring the N501Y mutations is needed. [ABSTRACT FROM AUTHOR]

Background The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. Methods Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. Results PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. Conclusions Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria. [ABSTRACT FROM AUTHOR]

Background: Severe acute malnutrition (SAM) is a major public health problem affecting children under the age of five in many low- and middle-income countries, and its resolution would contribute towards achieving the several sustainable development goals. The etiology of SAM is pluri-factorial, including delayed maturation of the gut microbiota, suboptimal feeding practices and dysfunctional breastfeeding. The recent serendipitous detection of Listeria monocytogenes in the breast milk of Malian women, in contrast to French women, suggests a possible association with SAM. Methodology/ Principal findings: To investigate the possible association of L. monocytogenes carriage in breast milk and SAM, a case-control study was performed in Senegal, with subjects recruited from two areas. Using 16S amplicon sequencing, a culture independent method, 100% (152/152) of the mothers were positive for L. monocytogenes in their breast milk while qPCR analysis gave lower recovery rates. Interestingly, after enrichment in Fraser broth and seeding on PALCALM agar, all 10 isolated strains were isolated from the milk of 10 mothers who had SAM children which also had a significantly increased relative abundance of L. monocytogenes (0.34 (SD 0.35) vs 0.05 (SD 0.07) in controls, p<0.0001). The high genomic similarity between these strains and Malian breast milk strains from a previous study supports the hypothesis of endemic clone carriage in West Africa. Moreover, the in vitro growth inhibition of L. monocytogenes using breast milk samples was obtained from only 50% of the milk of mothers who had SAM children, in contrast to control samples which systematically inhibited the growth of L. monocytogenes with a higher inhibition diameter (15.7 mm (SD 2.3) in controls versus 3.5 mm (SD 4.6) in SAM, p = 0.0001). Lactobacillus and Streptococcus isolated from the breast milk of controls inhibit L. monocytogenes in a species-dependent manner. Conclusions/Significance: Our study reveals a previously unsuspected carriage of L. monocytogenes in the breast milk of West African women, which is associated with SAM. The inhibitory effect of human selected lactic acid bacterial species against L. monocytogenes might provide new therapeutic and inexpensive options to prevent and treat this neglected public health issue. Author summary: Severe acute malnutrition is a global public health issue which greatly impacts childhood mortality rates. Although still not fully understood, the multi-factorial pathology of severe acute malnutrition has been associated, among other factors, with sub-optimal feeding practices (including dysfunctional breastfeeding) and an altered gut microbiota. The serendipitous detection of Listeria monocytogenes in the breast milk of Malian women has raised the possibility of its involvement in the pathogenesis of severe acute malnutrition. To investigate this possibility, the presence of L. monocytogenes was assessed in a cohort of lactating Senegalese women, both mothers of healthy children as well as those of severely malnourished children using culture-dependent and independent methods. Our study confirms the previously unsuspected presence of L. monocytogenes in the breast milk of Senegalese women, which is increased in the milk of mothers of severely malnourished children. Moreover, breast milk samples from the mothers of healthy children more frequently induced a potent inhibition of L. monocytogenes than those from the mothers of severely malnourished children. An inhibition was also achieved using potential probiotics, Lactobacillus and Streptococcus species, isolated from breast milk. Our study reveals the previously unsuspected carriage of L. monocytogenes in the breast milk of West African women, which is associated with severe acute malnutrition. The inhibitory effect of human selected lactic acid bacterial species against L. monocytogenes might provide new therapeutic and inexpensive options to prevent and treat this neglected public health issue. [ABSTRACT FROM AUTHOR]

This document is a retraction note from the journal Scientific Reports. The article in question, titled "Dramatic HIV DNA degradation associated with spontaneous HIV suppression and disease-free outcome in a young seropositive woman following her infection," has been retracted due to concerns about consent to participate. The authors were unable to provide documentation confirming that the patients who provided the samples used in the study gave consent for research purposes. The retraction is not agreed upon by all authors. [Extracted from the article]

Few publications, often limited to one specific pathogen, have studied bonobos (Pan paniscus), our closest living relatives, as possible reservoirs of certain human infectious agents. Here, 91 stool samples from semicaptive bonobos and bonobos reintroduced in the wild, in the Democratic Republic of the Congo, were screened for different infectious agents: viruses, bacteria and parasites. We showed the presence of potentially zoonotic viral, bacterial or parasitic agents in stool samples, sometimes coinfecting the same individuals. A high prevalence of Human mastadenoviruses (HAdV-C, HAdV-B, HAdV-E) was observed. Encephalomyocarditis viruses were identified in semicaptive bonobos, although identified genotypes were different from those identified in the previous fatal myocarditis epidemic at the same site in 2009. Non-pallidum Treponema spp. including symbiotic T. succinifaciens, T. berlinense and several potential new species with unknown pathogenicity were identified. We detected DNA of non-tuberculosis Mycobacterium spp., Acinetobacter spp., Salmonella spp. as well as pathogenic Leptospira interrogans. Zoonotic parasites such as Taenia solium and Strongyloides stercoralis were predominantly present in wild bonobos, while Giardia lamblia was found only in bonobos in contact with humans, suggesting a possible exchange. One third of bonobos carried Oesophagostomum spp., particularly zoonotic O. stephanostomum and O. bifurcum-like species, as well as other uncharacterized Nematoda. Trypanosoma theileri has been identified in semicaptive bonobos. Pathogens typically known to be transmitted sexually were not identified. We present here the results of a reasonably-sized screening study detecting DNA/RNA sequence evidence of potentially pathogenic viruses and microorganisms in bonobo based on a noninvasive sampling method (feces) and focused PCR diagnostics. [ABSTRACT FROM AUTHOR]

To date there are thirteen species validly assigned to the genus Anaerococcus. Most of the species in this genus are anaerobic and of human origin. Anaerococcus urinimassiliensis sp. nov., strain Marseille-P2143T is member of family Peptoniphilaceae, which was isolated from the urine of a 17-year-old boy affected by autoimmune hepatitis and membranoproliferative glomerulonephritis using the culturomic approach. In the current study, a taxono-genomics method was employed to describe this new species. The strain Marseille-P2143T was gram positive cocci with translucent colonies on blood agar. Its genome was 2,189,509 bp long with a 33.5 mol% G + C content and exhibited 98.48% 16S rRNA similarity with Anaerococcus provencensis strain 9,402,080. When Anaerococcus urinomassiliensis strain Marseill-P2143T is compared with closely related species, the values ranged from 71.23% with A. hydrogenalis strain DSM 7454T (NZ_ABXA01000052.1) to 90.64% with A. provencensis strain 9402080T (NZ_HG003688.1). This strain has implemented the repertoire of known bacteria of the human urinary tract. [ABSTRACT FROM AUTHOR]

Over the past decade, metagenomics has become the preferred method for exploring complex microbiota such as human gut microbiota. However, several bias affecting the results of microbiota composition, such as those due to DNA extraction, have been reported. These bias have been confirmed with the development of culturomics technique. In the present study, we report the contamination of a gnotobiotic mice unit with a bacterium first detected by gram staining. Scanning electron microscopy and transmission electron microscopy permitted to detect a bacterium with a thick cell wall. However, in parallel, the first attempt to identify and culture this bacterium by gene amplification and metagenomics of universal 16S rRNA failed. Finally, the isolation in culture of a fastidious bacterium not detected by using universal PCR was successfully achieved by using a BCYE agar plate with CO2 atmosphere at 30 °C. We performed genome sequencing of this bacterium using a strong extraction procedure. The genomic comparison allowed us to classify this bacterium as Klenkia terrae. And finally, it was also detected in the stool and kibble that caused the contamination by using specific qPCR against this bacterium. The elucidation of this contamination provides additional evidence that DNA extraction could be a bias for the study of the microbiota. Currently, most studies that strive to analyze and compare the gut microbiota are based on metagenomics. In a gnotobiotic mice unit contaminated with the fastidious Actinobacteria Klenkia terrae, standard culture, 16S rRNA gene amplification and metagenomics failed to identify the micro-organism observed in stools by gram-staining. Only a procedure based on culturomics allowed us to identify this bacterium and to elucidate the mode of contamination of the gnotobiotic mice unit through diet. [ABSTRACT FROM AUTHOR]