Single-Cell Growth Probability of Listeria monocytogenes at Suboptimal Temperature, pH, and Water Activity

The single-cell growth probability of Listeria monocytogenes was characterized in tryptone soy broth supplemented with 0.6% yeast extract at temperature ranging from 5 to 25°C, pH ranging from 4.4 to 6.5, and water activity ranging from 0.919 to 0.989. Growth was monitored visually in 96-microwell plates and the growth probability was deduced from concentrations estimated with the MPN calculation. Models were proposed to describe the increase of the probability from 0 to 1 with increasing values of environmental factors. An exponential model was used for the temperature and the individual minimal temperature for growth ranged from −3.6°C [−4.5°C, −2.7°C] to 17.3°C [16.0°C, 18.6°C]. An inverse exponential model was convenient for the pH and the minimal pH for growth of individual cells ranged from 4.34 [3.93, 4.75] to 5.93 [4.85, 7.01]. A linear model was used for the water activity and the minimal value for growth of individual cells ranged from 0.917 [0.909, 0.925] to 0.988 [0.966, 1]. In spite of the great between-experiment variability, the minimal values estimated for the growth limits of individual cells were in accordance with values usually proposed for L. monocytogenes populations. This study provides models allowing the prediction of the growth probability of a few cells contaminating chilled foods with suboptimal pH and water activity improving thus the assessment of the behavior of L. monocytogenes cells naturally contaminating foods

Paper microfluidics for nucleic acid amplification testing (NAAT) of infectious diseases

International audienceThe diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience

Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics

International audienceThe most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens

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

Nanopore sequencing of a monkeypox virus strain isolated from a pustular lesion in the Central African Republic

International audienceMonkeypox is an emerging and neglected zoonotic disease whose number of reported cases has been gradually increasing in Central Africa since 1980. This disease is caused by the monkeypox virus (MPXV), which belongs to the genus Orthopoxvirus in the family Poxviridae . Obtaining molecular data is particularly useful for establishing the relationships between the viral strains involved in outbreaks in countries affected by this disease. In this study, we evaluated the use of the MinION real-time sequencer as well as different polishing tools on MinION-sequenced genome for sequencing the MPXV genome originating from a pustular lesion in the context of an epidemic in a remote area of the Central African Republic. The reads corresponding to the MPXV genome were identified using two taxonomic classifiers, Kraken2 and Kaiju. Assembly of these reads led to a complete sequence of 196,956 bases, which is 6322 bases longer than the sequence previously obtained with Illumina sequencing from the same sample. The comparison of the two sequences showed mainly indels at the homopolymeric regions. However, the combined use of Canu with specific polishing tools such as Medaka and Homopolish was the best combination that reduced their numbers without adding mismatches. Although MinION sequencing is known to introduce a number of characteristic errors compared to Illumina sequencing, the new polishing tools allow a better-quality MinION-sequenced genome, thus to be used to help determine strain origin through phylogenetic analysis

First detection and genome sequencing of SARS‐CoV‐2 in an infected cat in France

International audienceAfter its first description in Wuhan (China), SARS-CoV-2 the agent of coronavirus disease 2019 (COVID-19) rapidly spread worldwide. Previous studies suggested that pets could be susceptible to SARS-CoV-2. Here, we investigated the putative infection by SARS-CoV-2 in 22 cats and 11 dogs from owners previously infected or suspected of being infected by SARS-CoV-2. For each animal, rectal, nasopharyngeal swabs and serum were taken. Swabs were submitted to RT-qPCR assays targeting 2 genes of SARS-CoV-2. All dogs were tested SARS-CoV-2 negative. One cat was tested positive by RT-qPCR on rectal swab. Nasopharyngeal swabs from this animal were tested negative. This cat showed mild respiratory and digestive signs. Serological analysis confirms the presence of antibodies against the SARS-CoV-2 in both serum samples taken 10 days apart. Genome sequence analysis revealed that the cat SARS-CoV-2 belongs to the phylogenetic clade A2a like most of the French human SARS-CoV-2. This study reports for the first time the natural infection of a cat in France (near Paris) probably through their owners. There is currently no evidence that cats can spread COVID-19 and owners should not abandon their pets or compromise their welfare

Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings

International audienceObjective: A massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-limited countries. We have developed and evaluated the loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by the WHO (≥20 000 and ≥ 200 000 IU/mL).Methods: Pan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively.Results: Our primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40 and 400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI 0.93-0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20 000 IU/mL; and 0.98 (0.97-0.99), 98.0% and 94.6% for ≥200 000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93-0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200 000 IU/mL. The assay was not adapted to discriminate low-level viraemia.Discussion: We have developed a simple, rapid (60 min), and inexpensive (US$8/assay) alternative to PCR to diagnose high viraemia ≥200 000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries

Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa

International audienceBackground: In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide. Methods: Whole genome sequencing was performed using NGS approach on six SARS. CoV-2 highly positive samples detected during the early phase of the outbreak. Results: Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development. Conclusions: These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level