Genomic characterization and phylogenetic analysis of the first SARS-CoV-2 variants introduced in Lebanon

International audienceBackgroundIn December 2019, the COVID-19 pandemic initially erupted from a cluster of pneumonia cases of unknown origin in the city of Wuhan, China. Presently, it has almost reached 94 million cases worldwide. Lebanon on the brink of economic collapse and its healthcare system thrown into turmoil, has previously managed to cope with the initial SARS-CoV-2 wave. In this study, we sequenced 11 viral genomes from positive cases isolated between 2 February 2020 and 15 March 2020.MethodsSequencing data was quality controlled, consensus sequences generated, and a maximum-likelihood tree was generated with IQTREE v2. Genetic lineages were assigned with Pangolin v1.1.14 and single nucleotide variants (SNVs) were called from read files and manually curated from consensus sequence alignment through JalView v2.11 and the genomic mutational interference with molecular diagnostic tools was assessed with the CoV-GLUE pipeline. Phylogenetic analysis of whole genome sequences confirmed a multiple introduction scenario due to international travel.ResultsThree major lineages were identified to be circulating in Lebanon in the studied period. The B.1 (20A clade) was the most prominent, followed by the B.4 lineage (19A clade) and the B.1.1 lineage (20B clade). SNV analysis showed 15 novel mutations from which only one was observed in the spike region

Outer Membrane Proteome of Veillonella parvula: A Diderm Firmicute of the Human Microbiome

International audienceVeillonella parvula is a biofilm-forming commensal found in the lungs, vagina, mouth, and gastro-intestinal tract of humans, yet it may develop into an opportunistic pathogen. Furthermore, the presence of Veillonella has been associated with the development of a healthy immune system in infants. Veillonella belongs to the Negativicutes, a diverse clade of bacteria that represent an evolutionary enigma: they phylogenetically belong to Gram-positive (monoderm) Firmicutes yet maintain an outer membrane (OM) with lipopolysaccharide similar to classic Gram-negative (diderm) bacteria. The OMs of Negativicutes have unique characteristics including the replacement of Braun's lipoprotein by OmpM for tethering the OM to the peptidoglycan. Through phylogenomic analysis, we have recently provided bioinformatic annotation of the Negativicutes diderm cell envelope. We showed that it is a unique type of envelope that was present in the ancestor of present-day Firmicutes and lost multiple times independently in this phylum, giving rise to the monoderm architecture; however, little experimental data is presently available for any Negativicutes cell envelope. Here, we performed the first experimental proteomic characterization of the cell envelope of a diderm Firmicute, producing an OM proteome of V. parvula. We initially conducted a thorough bioinformatics analysis of all 1,844 predicted proteins from V. parvula DSM 2008's genome using 12 different localization prediction programs. These results were complemented by protein extraction with surface exposed (SE) protein tags and by subcellular fractionation, both of which were analyzed by liquid chromatography tandem mass spectrometry. The merging of proteomics and bioinformatics results allowed identification of 78 OM proteins. These include a number of receptors for TonB-dependent transport, the main component of the BAM system for OM protein biogenesis (BamA), the Lpt system component LptD, which is responsible for insertion of LPS into the OM, and several copies of the major OmpM protein. The annotation of V. parvula's OM proteome markedly extends previous inferences on the nature of the cell envelope of Negativicutes, including the experimental evidence of a BAM/TAM system for OM protein biogenesis and of a complete Lpt syste

MEMHDX: An interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

International audienceMotivation: With the continued improvement of requisite mass spectrometers and UHPLC systems, Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) workflows are rapidly evolving towards the investigation of more challenging biological systems, including large protein complexes and membrane proteins. The analysis of such extensive systems results in very large HDX-MS datasets for which specific analysis tools are required to speed up data validation and interpretation. Results: We introduce a web application and a new R-package named " MEMHDX " to help users analyze, validate and visualize large HDX-MS datasets. MEMHDX is composed of two elements. A statistical tool aids in the validation of the results by applying a mixed-effects model for each peptide, in each experimental condition, and at each time point, taking into account the time dependency of the HDX reaction and number of independent replicates. Two adjusted p-values are generated per peptide, one for the " Change in dynamics " and one for the " Magnitude of ∆D " , and are used to classify the data by means of a " Logit " representation. A user-friendly interface developed with Shiny by RStudio facilitates the use of the package. This interactive tool allows the user to easily and rapidly validate, visualize and compare the relative deuterium incorporation on the amino acid sequence and 3D structure, providing both spatial and temporal information. Availability: MEMHDX is freely available as a web tool at the project home page http://memhdx

Rapid Genomic Characterization of SARS-CoV-2 by Direct Amplicon-Based Sequencing Through Comparison of MinION and Illumina iSeq100TM System

International audienceGlobal human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with coronavirus-related human diseases, such as the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. Here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held MinION sequencer and the latest most compact Illumina sequencer, the iSeq100TM system. Our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of SARS-CoV-2 from clinical samples in just a few hours. Both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas

Automated Phosphopeptide Identification Using Multiple MS/MS Fragmentation Modes

International audiencePhosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, I:: and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana. We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis.

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection

International audienceAt the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBβ-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNβ, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling

Third Generation Sequencing Technologies to Decipher Genomic Structures of Recombinant-prone Viruses

International audienceINTRODUCTION/CONTEXT The development of the “third-generation sequencing” platforms, such as the Pacific Biosciences PacBio sequencing system and more recently the Oxford Nanopore MinION device, have yet to be exploited, and have generated particular interest within the scientific community. These methodologies open up new possibilities, such as providing the capability for minimal library preparation and long reads (up to 10 kilobases), thus enabling true linkage to be established between variants within single genomes, and resolving assembly issues that often give incorrect genomic organization.These long-read sequencing platforms especially facilitate the analysis of viral genome structure, including recombination events, which are generally difficult to ascertain using second-generation platforms such as Illumina and Ion Torrent. In fact, current second-generation sequencing technologies have played a driving role to address questions relating to viral genome organization, epidemiology, and investigations of outbreaks by characterizing both partial- (such structural proteins) and whole-genome sequencing (WGS). In the specific case of recombinant-prone viruses, e.g. members of Picornaviridae family, second-generation sequencing technologies have often unveiled the limits of the approach, notably when determining precise viral genomic reconstruction and recombination hotspots. Foot-and-mouth disease (FMD) is considered one of the most contagious diseases of livestock, which can lead to huge economic losses. This disease, present in Africa, Asia and South America, is caused by a virus from the Picornaviridae family, genus Aphthovirus, referred to as FMD virus (FMDV). Seven different FMDV serotypes have been described (A, O, C, SAT1, SAT2, SAT3 and Asia1). The genome of FMDV comprises a positive-sense single-stranded RNA approximately 8300 nucleotides in length. The viral genome contains a single long ORF, encoding a large polyprotein, further processed into 13 viral mature proteins, whose 4 structural proteins (VP1-VP4). The extensive genetic diversity in FMDV is attributed to the poor proof-reading ability of the viral RNA dependent RNA polymerase, with large viral population size and high replication rates. Then, FMDV evolves through genetic drift, where positive selection contributes to fixation of mutations in the capsid coding regions. Although the VP1 coding region of FMDV is useful for isolate characterization, it is relatively short (only ~8% of the genome length) and, consequently, phylogenetic trees generated from closely related FMDV sequences recovered within outbreak clusters are typically flat, with poor resolution. For this reason, the use of WGS to discriminate between closely related viruses has become commonplace and has subsequently been applied to both human and animal pathogens. However, incongruences between phylogenies from individual sub-genomic regions suggest that recombination also plays a role in FMDV evolution. Recombination events have indeed been demonstrated within the FMDV genome and have highlighted the fact that particular regions of the FMDV genome appear to be more prone to intertypic recombination than others. The number of exchanges of genome sequences encoding for nonstructural proteins seems to be much more important and numerous, than the events involving the sequences encoding parts of the capsid-coding region. It is therefore important to identify the set of recombination events in FMDV full genome sequences, and to determine the distribution of these events across the FMDV genome. Recombination events are of particular interest as a source for driving FMDV diversity giving rise to FMDV outbreaks. Third-generation sequencing technologies could thus allow to bridge the gap in resolving genome structure uncertainties for such virus.METHODSFour isolates of FMDV were sequenced using MiSeq Illumina platform (second-generation) and MinION Oxford Nanopore Technologies (third-generation). Two of these samples were collected from cattle in 2011 from Balochistan Province in Pakistan (PAK-6; PAK-9) and the others originated from Benin (BEN-017, BEN-036) in 2010. The whole genome sequencing (WGS) with Illumina technology were performed using Nextera XT kit in order to produce paired-end reads of approximately 150pb each. The MinION libraries were prepared using 1D2 Sequencing chemistry and Flow cell MIN-10 to obtain one unique long read covering the entire genome of the virus (8Kb). For second-generation data analysis, the four FMDV genomes were reconstructed using a dedicated pipeline with classic state-of-the-art bioinformatics tools. Third-generation long reads were analyzed using a long reads analysis workflow (including Albacore and Canu Minimap softwares). In both approaches, phylogenetic trees were established using the Mafft tool, allowing to consolidate the geographical origin and the serotype of all isolates and to help solve the recombination events.A global genomics analysis approach for mapping recombination hotspots appeared to be necessary, particularly for such datasets where the identities of the parental sequences involved in recombination are unknown. More specifically, within the current data study-set, it is generally unknown which FMDV sequence is the recombinant and which is no recombinant. Mapping of the positions of recombination is done by a phylogenetic-compatibility analysis using phylogeny tree scanning, applied to both publicly available full genomes and newly sequenced isolates. Phylogenetic tree scanning is based on recording the order of each variant in an alignment, giving a successive serie of phylogenetic trees (rooted neighbor-joining trees, 100 bootstrap replicates, and where all branches with <70% support are collapsed, moving windows of 300nt and intervals of 100nt), and hence examining the positions in the alignment where phylogenetic relationships change. To investigate the extent of recombination within the data set, the aligned sequences were examined using the Recombination Detection Program in RDP4, in order to infer breakpoint positions and recombinant sequences for every detected potential recombination event. The results of this analysis are in agreement with the phylogenetic-compatibility analysis in that the distribution of observed breakpoints appears to be non-random.CONCLUSION. Incongruent tree topologies between the structural and non-structural coding regions of FMDV isolates suggest that the VP1 phylogeny may not be appropriately reflecting the evolutionary histories of different FMDV isolates. We therefore analyzed the existence of differences in the frequency of recombination between species by an extended comparison of sequences that included all available complete genome sequences available from public databases. Using exhaustive comparisons of fragment sets generated from alignments or the complete genome sequence of the species, it is possible to map regions of phylogenetic incongruity and infer sites of favored recombination using a phylogenetic compatibility matrix (PCM). The results of these FMDV breakpoint distribution and phylogenetic-compatibility analyses reflect a clear partitioning of structural and non-structural genes in the organization of the genome. This organization facilitates component swapping or recombination that frequently occurs among such viruses. Confident construction of transmission trees from phylogenetic data, through spatio-temporal epidemiological data, using MinION nanopore sequencing, offers an exciting potential to FMDV diagnostics, and more specifically for resolving recombination scenarios when comparing different field isolates. Such approaches, integrating both novel technological sequencing instruments, together with phylogenetic and epidemiological data, will help understand mechanisms involving the recombination patterns observed in FMDV and other picornaviruses, and will eventually lead to novel insights into epidemiological and phylogeographics issues in FMDV outbreaks