Whole-genome, deep pyrosequencing analysis of a duck influenza A virus evolution in swine cells.

We studied the sub-population level evolution of a duck influenza A virus isolate during passage in swine tracheal cells. The complete genomes of the A/mallard/Netherlands/10-Nmkt/1999 strain and its swine cell-passaged descendent were analysed by 454 pyrosequencing with coverage depth ranging from several hundred to several thousand reads at any point. This allowed characterization of defined minority sub-populations of gene segments 2, 3, 4, 5, 7, and 8 present in the original isolate. These minority sub-populations ranged between 9.5% (for segment 2) and 46% (for segment 4) of their respective gene segments in the parental stock. They were likely contributed by one or more viruses circulating within the same area, at the same period and in the same or a sympatric host species. The minority sub-populations of segments 3, 4, and 5 became extinct upon viral passage in swine cells, whereas the minority sub-populations of segments 2, 7 and 8 completely replaced their majority counterparts. The swine cell-passaged virus was therefore a three-segment reassortant and also harboured point mutations in segments 3 and 4. The passaged virus was more homogenous than the parental stock, with only 17 minority single nucleotide polymorphisms present above 5% frequency across the whole genome. Though limited here to one sample, this deep sequencing approach highlights the evolutionary versatility of influenza viruses whereby they exploit their genetic diversity, predilection for mixed infection and reassortment to adapt to a new host environmental niche.This work was supported by a grant from DEFRA and HEFCE under the Veterinary Training and Research Initiative to the Cambridge Infectious Diseases Consortium (VB, LT), BBSRC grants BB/H014306/1 and BB/G00479X/1 (LT), and the French Ministry of Agriculture, INRA and the French Région Midi-Pyrénées (GC, J-LG, VB).This is the accepted version of the original version available at: http://dx.doi.org/10.1016/j.meegid.2013.04.03

An amplicon-based nanopore sequencing workflow for rapid tracking of avian influenza outbreaks, France, 2020-2022

During the recent avian influenza epizootics that occurred in France in 2020/21 and 2021/22, the virus was so contagiousness that it was impossible to control its spread between farms. The preventive slaughter of millions of birds consequently was the only solution available. In an effort to better understand the spread of avian influenza viruses (AIVs) in a rapid and innovative manner, we established an amplicon-based MinION sequencing workflow for the rapid genetic typing of circulating AIV strains. An amplicon-based MinION sequencing workflow based on a set of PCR primers targeting primarily the hemagglutinin gene but also the entire influenza virus genome was developed. Thirty field samples from H5 HPAIV outbreaks in France, including environmental samples, were sequenced using the MinION MK1C. A real-time alignment of the sequences with MinKNOW software allowed the sequencing run to be stopped as soon as enough data were generated. The consensus sequences were then generated and a phylogenetic analysis was conducted to establish links between the outbreaks. The whole sequence of the hemagglutinin gene was obtained for the 30 clinical samples of H5Nx HPAIV belonging to clade 2.3.4.4b. The consensus sequences comparison and the phylogenetic analysis demonstrated links between some outbreaks. While several studies have shown the advantages of MinION for avian influenza virus sequencing, this workflow has been applied exclusively to clinical field samples, without any amplification step on cell cultures or embryonated eggs. As this type of testing pipeline requires only a short amount of time to link outbreaks or demonstrate a new introduction, it could be applied to the real-time management of viral epizootics

Highly multiplexed quantitative PCR-based platform for evaluation of chicken immune responses

To address the need for sensitive high-throughput assays to analyse avian innate and adaptive immune responses, we developed and validated a highly multiplexed qPCR 96.96 Fluidigm Dynamic Array to analyse the transcription of chicken immune-related genes. This microfluidic system permits the simultaneous analysis of expression of 96 transcripts in 96 samples in 6 nanolitre reactions and the 9,216 reactions are ready for interpretation immediately. A panel of 89 genes was selected from an RNA-seq analysis of the transcriptional response of chicken macrophages, dendritic cells and heterophils to agonists of innate immunity and from published transcriptome data. Assays were confirmed to be highly specific by amplicon sequencing and melting curve analysis and the reverse transcription and preamplification steps were optimised. The array was applied to RNA of various tissues from a commercial line of broiler chickens housed at two different levels of biosecurity. Gut-associated lymphoid tissues, bursa, spleen and peripheral blood leukocytes were isolated and transcript levels for immune-related genes were defined. The results identified blood cells as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially transcribed between birds housed under varying biosecurity levels. Conventional qPCR analysis of three differentially transcribed genes confirmed the results from the multiplex qPCR array. A highly multiplexed qPCR-based platform for evaluation of chicken immune responses has been optimised and validated using samples from commercial chickens. Apart from applications in selective breeding programmes, the array could be used to analyse the complex interplay between the avian immune system and pathogens by including pathogen-specific probes, to screen vaccine responses, and as a predictive tool for immune robustness

Analyse de la variabilité génétique des virus influenza aviaires par séquençage à très haut débit.

Les virus influenza A sont des virus à ARN segmenté et présentent une importante variabilité génétique. Les virus influenza aviaires (VIA) peuvent être transmis depuis leur réservoir (les oiseaux aquatiques sauvages) vers les volailles domestiques ou les mammifères chez qui ils peuvent causer des épizooties avec un fort potentiel de pertes économiques et une menace pour la santé humaine.Le suivi virologique des VIA lors d’épisodes infectieux est primordial pour la détection précoce de modifications génétiques. Le développement de nouvelles générations de séquençage (NGS) permet d’effectuer un suivi de l’évolution génétique virale de façon pratique et accessible.Ce document décrit l’application du pyroséquençage 454 de Roche (i) à l’analyse de l’adaptation d’un VIA de sous-type H6N1 d’un élevage de canards vers un élevage de dindes lors d’un épisode infectieux de terrain et (ii) au suivi de l’adaptation d’un VIA de sous-type H1N1 à des cellules porcines.Dans le contexte de l’évolution du VIA de sous-type H6N1, une troncation du gène de la neuraminidase (NA) a été mise en évidence par pyroséquençage dans moins de 2 % des échantillons de canards alors qu’elle était présente dans 100 % des échantillons de dindes. Cela suggère que la délétion de la NA permet l’adaptation des VIA aux volailles et peut émerger suite à une modification de la pression de sélection.Lors du suivi de l’adaptation d’un VIA de sous-type H1N1 à des cellules de trachée de porcelet, les génomes complets des virus parental et adapté ont été analysés par pyroséquençage. La couverture et la profondeur de séquençage ont permis la caractérisation de populations virales minoritaires dans l’échantillon parental qui ont émergé après l’adaptation de l’échantillon viral sur cellules porcines.Ces deux études démontrent à quel point le séquençage à très haut débit met en évidence les fortes capacités d’évolution des VIA qui leur permettent de s’adapter à de nouvelles niches environnementales

Next generation sequencing and high-throughput sequencing : Application to detection and characterization of avian respiratory pathogens and to the control of vaccine purity

La capacité de détection des agents pathogènes est un enjeu croissant tant les maladies infectieuses représentent un risque pour la santé animale et humaine. La globalisation des échanges commerciaux et des voyages, l’évolution des pratiques agricoles, les changements climatiques ou encore les migrations de masse sont autant de facteurs bouleversant la biologie des micro-organismes et de fait, leurs capacités d’émergence. Ce manuscrit décrit trois approches complémentaires, basées sur trois techniques innovantes de biologie moléculaire pour la détection d’agents pathogènes et appliquées à trois contextes différents : (i) la recherche d’une liste précise de micro-organismes par PCR quantitative en temps réel en format microfluidique, (ii) la détection sans a priori d’agents infectieux dans un milieu complexe par métagénomique et séquençage Illumina (Miseq) et (iii) le génotypage d’un agent infectieux sans amplification préalable des génomes par NGS (Nouvelles Générations de séquençage) de troisième génération, le MinION d’Oxford Nanopore Technologies. Ces trois études ont permis de montrer l’apport de ces techniques, qui présentent toutes des caractéristiques distinctes, adaptées à différentes applications. Au-delà de l’application de ces techniques au domaine du diagnostic microbiologique, leur utilisation dans le cadre du contrôle des médicaments immunologiques vétérinaires est une perspective prioritaire de ce travail. En effet, les préparations vaccinales vétérinaires sont soumises à l’obligation de recherche d’une liste d’agents pathogènes à exclure mais également à la vérification de l’identité génétique des souches vaccinales. L’accessibilité et les performances exponentielles des nouvelles technologies de PCR et de séquençage ouvrent ainsi des perspectives révolutionnaires dans le domaine du diagnostic et du contrôle microbiologique.Detection of pathogens becomes an increasing challenge, since infectious diseases represent major risks for both human and animal health. Globalization of trade and travels, evolution of farming practices and global climatic changes, as well as mass migrations are impacting the biology of pathogens and their emerging potential. This manuscript describes three approaches, based on three innovative technologies of molecular biology applied to the detection of pathogens in three different settings : (i) detection of a list of pathogens using real-time quantitative PCR on a microfluidic platform, (ii) unbiased detection of pathogens in complex matrix, using metagenomics and Illumina (Miseq) sequencing and (iii) genotyping of pathogens without isolation of PCR-enrichment using a 3rd generation NGS (Next Generation Sequencing) platform MinION from Oxford Nanopore Technologies. The three studies shown the contribution of these techniques, each representing distinctive features, suitable for the respective applications. Beyond application of these techniques to the field of microbial diagnostics, their use for the control of veterinary immunological drugs is a priority of this project. Veterinary vaccines are not only submitted to mandatory detection of listed pathogens to be excluded, but also to validation of the genetic identity of vaccine strains. The exponential availability and performances of new PCR or sequencing technologies open cutting-edge perspectives in the field of microbial diagnostic and control

Séquençage et PCR à haut débit : application à la détection et la caractérisation d'agents pathogènes respiratoires aviaires et au contrôle de pureté microbiologique des vaccins

Detection of pathogens becomes an increasing challenge, since infectious diseases represent major risks for both human and animal health. Globalization of trade and travels, evolution of farming practices and global climatic changes, as well as mass migrations are impacting the biology of pathogens and their emerging potential. This manuscript describes three approaches, based on three innovative technologies of molecular biology applied to the detection of pathogens in three different settings : (i) detection of a list of pathogens using real-time quantitative PCR on a microfluidic platform, (ii) unbiased detection of pathogens in complex matrix, using metagenomics and Illumina (Miseq) sequencing and (iii) genotyping of pathogens without isolation of PCR-enrichment using a 3rd generation NGS (Next Generation Sequencing) platform MinION from Oxford Nanopore Technologies. The three studies shown the contribution of these techniques, each representing distinctive features, suitable for the respective applications. Beyond application of these techniques to the field of microbial diagnostics, their use for the control of veterinary immunological drugs is a priority of this project. Veterinary vaccines are not only submitted to mandatory detection of listed pathogens to be excluded, but also to validation of the genetic identity of vaccine strains. The exponential availability and performances of new PCR or sequencing technologies open cutting-edge perspectives in the field of microbial diagnostic and control.La capacité de détection des agents pathogènes est un enjeu croissant tant les maladies infectieuses représentent un risque pour la santé animale et humaine. La globalisation des échanges commerciaux et des voyages, l’évolution des pratiques agricoles, les changements climatiques ou encore les migrations de masse sont autant de facteurs bouleversant la biologie des micro-organismes et de fait, leurs capacités d’émergence. Ce manuscrit décrit trois approches complémentaires, basées sur trois techniques innovantes de biologie moléculaire pour la détection d’agents pathogènes et appliquées à trois contextes différents : (i) la recherche d’une liste précise de micro-organismes par PCR quantitative en temps réel en format microfluidique, (ii) la détection sans a priori d’agents infectieux dans un milieu complexe par métagénomique et séquençage Illumina (Miseq) et (iii) le génotypage d’un agent infectieux sans amplification préalable des génomes par NGS (Nouvelles Générations de séquençage) de troisième génération, le MinION d’Oxford Nanopore Technologies. Ces trois études ont permis de montrer l’apport de ces techniques, qui présentent toutes des caractéristiques distinctes, adaptées à différentes applications. Au-delà de l’application de ces techniques au domaine du diagnostic microbiologique, leur utilisation dans le cadre du contrôle des médicaments immunologiques vétérinaires est une perspective prioritaire de ce travail. En effet, les préparations vaccinales vétérinaires sont soumises à l’obligation de recherche d’une liste d’agents pathogènes à exclure mais également à la vérification de l’identité génétique des souches vaccinales. L’accessibilité et les performances exponentielles des nouvelles technologies de PCR et de séquençage ouvrent ainsi des perspectives révolutionnaires dans le domaine du diagnostic et du contrôle microbiologique

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020–2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.</p

Molecular detection and characterization of duck respiratory viruses using unbiased high-throughput sequencing

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Identificazione mediante Next Generation Sequencing di sottopopolazioni virali in un vaccino vivo attenuato per Metapneumovirus aviare sottotipo B e loro implicazione nel fenomeno di reversione a virulenza

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine and progenitor consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence were not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, cannot totally discounted from being involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release