Development of mobile laboratory for viral hemorrhagic fever detection in Africa

Background In order to enable local response to viral haemorrhagic fever outbreaks a mobile laboratory transportable on commercial flights was developed. Methodology The development progressed from use of mobile real time RT-PCR to mobile Recombinase Polymerase Amplification (RT-RPA). The various stages of the mobile laboratory development are described. Results A brief overview of its deployments, which culminated in the first on site detection of Ebola virus disease (EVD) in March 2014 and a successful use in a campaign to roll back EVD cases in Conakry in the West-Africa Ebola virus outbreak are described. Conclusion The developed mobile laboratory successfully enabled local teams to perform rapid viral haemorrhagic fever disgnostics

Avian influenza virus surveillance in high arctic breeding Geese, Greenland

International audienceThe connectedness in Arctic regions between migratory waterbird populations originating from different continents and the potential for virus exchange at their shared Arctic breeding ground point to the need to explore the largely unstudied circumpolar circulation of avian influenza viruses (AIV). We here report the investigation of AIV in wild birds and lakes in a high Arctic area of Northeast Greenland. No AIV could be detected in the fecal, feather, and water samples collected from large flocks of pink-footed geese Anser brachyrhynchus and barnacle geese Branta leucopsis in and around refuge lakes, where they congregate at high density during their flightless molting period in summer

Database of SARS-CoV-2 and coronaviruses kinetics relevant for assessing persistence in food processing plants

The authors are grateful to collaborators of the SACADA consortium: Géraldine Boué, Steven Duret, Michel Federighi, Maxence Feher, Lisa Fourniol, Yvonnick Guillois, Pauline Kooh, Sophie Le Poder, Anne-Laure Maillard, Jean-Claude Manuguerra, Mathilde Pivette, Jessica Vanhomwegen, Prunelle WaldmanInternational audienceSARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a virus causing severe acute respiratory disease in humans, emerged in late 2019. This respiratory virus can spread via aerosols, fomites, contaminated hands or surfaces as for other coronaviruses. Studying their persistence under different environmental conditions represents a key step for better understanding the virus transmission. This work aimed to present a reproducible procedure for collecting data of stability and inactivation kinetics from the scientific literature. The aim was to identify data useful for characterizing the persistence of viruses in the food production plants. As a result, a large dataset related to persistence on matrices or in liquid media under different environmental conditions is presented. This procedure, combining bibliographic survey, data digitalization techniques and predictive microbiological modelling, identified 65 research articles providing 455 coronaviruses kinetics. A ranking step as well as a technical validation with a Gage Repeatability & Reproducibility process were performed to check the quality of the kinetics. All data were deposited in public repositories for future uses by other researchers

Monkeypox virus phylogenetic similarities between a human case detected in Cameroon in 2018 and the 2017-2018 outbreak in Nigeria

International audienceA monkeypox virus was detected from a human clinical case in 2018 in Cameroon; a country where no human cases were reported since 1989. The virus exhibited close genetic relatedness with another monkeypox virus isolated in Nigeria during the 2017-2018 outbreak. Although our molecular findings argue in favor of an extension of the monkeypox outbreak from Nigeria into Cameroon, the possibility that the monkeypox virus detected could be indigenous to Cameroon cannot be ruled out

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

Molecular characterization of measles virus strains circulating in Cameroon during the 2013-2016 epidemics

International audienceThe first genotyping data on measles virus (MeV) strains in Cameroon dates from 1994, while other studies were realized in 2001 and 2011 with the establishment of MeV virological surveillance. However, the genetic data of MeV strains circulating in Cameroon remains fragmented and concentrated in certain regions, hence the need for an update. The objective of this study was to have recent data on MeV genotypes circulating in Cameroon. Ninety throat swabs collected during recent measles outbreaks were analyzed by MeV genotyping RT-PCR using the nucleoprotein gene N. The resulting sequences were analyzed on the basis of 450 nucleotides with MEGA 7 software. Overall genome analysis was performed on 40/90 sequences. The strains were from all ten regions and all belonged to cluster 1 of genotype B3. The genotype B3 has been circulating in Cameroon for long periods of time; efforts must be made in immunization for its elimination

ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

International audienceRT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro , suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population

Massively parallel pathogen identification using high-density microarrays

International audienceIdentification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole-genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1-4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner