Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish

A Six Years (2010–2016) Longitudinal Survey of the Four Serotypes of Dengue Viruses in Lao PDR

Dengue fever is the most prevalent arthropod-borne viral infection of humans in tropical and subtropical countries. Since 1979, dengue has been reported to be endemic in the Lao People’s Democratic Republic (PDR), as in many countries in Southeast Asia, with a complex circulation of the four dengue viruses’ serotypes (DENV-1 to DENV-4). By sequencing the complete envelope protein, we explored a panel of samples from five Lao Provinces (Vientiane capital, Luangprabang, Bolikhamxay, Saravane, Attapeu) to enrich knowledge about the co-circulation of DENVs in Lao PDR between 2010 and 2016. Phylogenetic analyses highlighted the specific circulation of DENV-1 genotype I, DENV-2 genotype Asian I, DENV-4 genotype I and the co-circulation of DENV-3 genotype II and III. The continuous co-circulation of the four serotypes was underlined, with genotype or cluster shifts among DENV-3 and DENV-1. These data suggested the emergence or re-emergence of DENV strains associated with epidemic events, potentially linked to the exchanges within the territory and with neighboring countries. Indeed, the increasing local or regional connections favored the dissemination of new isolates or new clusters around the country. Since 2012, the surveillance and alert system created in Vientiane capital by the Institut Pasteur du Laos appears to be a strategic tool for monitoring the circulation of the four serotypes, especially in this endemic country, and allows for improving dengue epidemiological knowledge to anticipate epidemic events better

Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae?▿

Comparative genomics analysis of the Tamil Nadu strain of Mycobacterium leprae has uncovered several polymorphic sites with potential as epidemiological tools. In this study we compared the stability of two different markers of genomic biodiversity of M. leprae in several biopsy samples isolated from the same leprosy patient. The first type comprises five different variable-number tandem repeats (VNTR), while the second is composed of three single nucleotide polymorphisms (SNP). Contrasting results were obtained, since no variation was seen in the SNP profiles of M. leprae from 42 patients from 7 different locations in Mali whereas the VNTR profiles varied considerably. Furthermore, since variation in the VNTR pattern was seen not only between different isolates of M. leprae but also between biopsy samples from the same patient, these VNTR may be too dynamic for use as epidemiological markers for leprosy

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

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYROT was isolated from a deep hypersaline anoxic basin called “Tyro” located in the Eastern Mediterranean Sea. Cells of SLHTYROT were motile cocci. The strain SLHTYROT grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L−1 NaCl (optimum 135 g L−1). Strain SLHTYROT was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYROT was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYROT was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYROT should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYROT (=DSM 108854 = JCM 32768 = UBOCC-M-3308)

Complete genome sequence of a vampire bat-related rabies virus obtained by metagenomics from a patient with encephalitis of unknown etiology, French Guiana

International audienceWe report the complete genome sequence of a rabies virus obtained by direct metagenomics from the cerebellum of a gold panner who died of unknown encephalitis in French Guiana. Phylogenetic analysis exhibited a close genetic relationship with vampire bat-related isolates, confirming the second case of human rabies identified in this territory

Using Background Sequencing Data to Anticipate DENV-1 Circulation in the Lao PDR

International audienceSince its first detection in 1979, dengue fever has been considered a major public health issue in the Lao People’s Democratic Republic (PDR). Dengue virus (DENV) serotype 1 was the cause of an epidemic in 2010–2011. Between 2012 and 2020, major outbreaks due successively to DENV-3, DENV-4 and recently DENV-2 have been recorded. However, DENV-1 still co-circulated in the country over this period. Here, we summarize epidemiological and molecular data of DENV-1 between 2016 and 2020 in the Lao PDR. Our data highlight the continuous circulation of DENV-1 in the country at levels ranging from 16% to 22% among serotyping tests. In addition, the phylogenetic analysis has revealed the circulation of DENV-1 genotype I at least since 2008 with a co-circulation of different clusters. Sequence data support independent DENV-1 introductions in the Lao PDR correlated with an active circulation of this serotype at the regional level in Southeast Asia. The maintenance of DENV-1 circulation over the last ten years supports a low level of immunity against this serotype within the Lao population. Thereby, the risk of a DENV-1 epidemic cannot be ruled out in the future, and this emphasizes the importance of maintaining an integrated surveillance approach to prevent major outbreak

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

Complete Genome Sequences of Monkeypox Virus from a French Clinical Sample and the Corresponding Isolated Strain, Obtained Using Nanopore Sequencing

International audienceWe report the whole-genome sequences of a monkeypox virus from the skin lesion of a French patient and the corresponding isolated viral strain. Both viral genomic sequences were successfully obtained by applying shotgun metagenomics using the Oxford Nanopore Technologies sequencing approach

Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen

International audienceWe report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample