Evaluation of three hemagglutinin-based vaccines for the experimental control of a panzootic clade 2.3.4.4b A(H5N8) high pathogenicity avian influenza virus in mule ducks.

In France during winter 2016-2017, 487 outbreaks of clade 2.3.4.4b H5N8 subtype high pathogenicity (HP) avian influenza A virus (AIV) infections were detected in poultry and captive birds. During this epizootic, HPAIV A/decoy duck/France/161105a/2016 (H5N8) was isolated and characterized in an experimental infection transmission model in conventional mule ducks. To investigate options to possibly protect such ducks against this HPAIV, three vaccines were evaluated in controlled conditions. The first experimental vaccine was derived from the hemagglutinin gene of another clade 2.3.4.4b A(H5N8) HPAIV. It was injected at three weeks of age, either alone (Vac1) or after a primer injection at day-old (Vac1 + boost). The second vaccine (Vac2) was a commercial bivalent adjuvanted vaccine containing an expressed hemagglutinin modified from a clade 2.3.2 A(H5N1) HPAIV. Vac2 was administered as a single injection at two weeks of age. The third experimental vaccine (Vac3) also incorporated a homologous 2.3.4.4b H5 HA gene and was administered as a single injection at three weeks of age. Ducks were challenged with HPAIV A/decoy duck/France/161105a/2016 (H5N8) at six weeks of age. Post-challenge virus excretion was monitored in vaccinated and control birds every 2-3 days for two weeks using real-time reverse-transcription polymerase chain reaction and serological analyses (haemagglutination inhibition test against H5N8, H5 ELISA and AIV ELISA) were performed. Vac1 abolished oropharyngeal and cloacal shedding to almost undetectable levels, whereas Vac3 abolished cloacal shedding only (while partially reducing respiratory shedding) and Vac2 only partly reduced the respiratory and intestinal excretion of the challenge virus. These results provided relevant insights in the immunogenicity of recombinant H5 vaccines in mule ducks, a rarely investigated hybrid between Pekin and Muscovy duck species that has played a critical role in the recent H5 HPAI epizootics in France.

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid and On-Site Detection of Avian Influenza Virus.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.

TIMP1 intron 3 retention is a marker of colon cancer progression controlled by hnRNPA1.

We previously reported a 40-transcripts signature marking the normal mucosa to colorectal adenocarcinoma transition. Eight of these mRNAs also showed splicing alterations, including a specific intron 3 retention in tissue metalloprotease inhibitor I (TIMP1), which decreased during the early steps of colorectal cancer progression. To decipher the mechanism of intron 3 retention/splicing, we first searched for putative RNA binding protein binding sites onto the TIMP1 sequence. We identified potential serine arginine rich splicing factor 1 (SRSF1) and heterogeneous nuclear RiboNucleoProtein A1 (hnRNPA1) binding sites at the end of intron 3 and the beginning of exon 4, respectively. RNA immunoprecipitation showed that hnRNPA1, but not SRSF1 could bind to the corresponding region in TIMP1 pre-mRNA in live cells. Furthermore, using a TIMP1-based ex vivo minigene approach, together with a plasmon resonance in vitro RNA binding assay, we confirmed that hnRNPA1 could indeed bind to wild type TIMP1 exon 4 pre-mRNA and control TMP1 intron 3 splicing, the interaction being abolished in presence of a mutant sequence that disrupted this site. These results indicated that hnRNPA1, upon binding to TIMP1 exon 4, was a positive regulator of intron 3 splicing. We propose that this TIMP1-intron 3 + transcript belongs to the class of nuclear transcripts with "detained" introns, an abundant molecular class, including in cancer.

[Pre-mRNA splicing: when the spliceosome loses ground]. / L'épissage des ARN pré-messagers : quand le splicéosome perd pied.

Pre-mRNA splicing is an obligatory step required to assemble the vast majority of mRNAs in eukaryotes. In humans, each gene gives rise to at least two transcripts, with an average 6-8 spliced transcripts per gene. Pre-mRNA splicing is not unequivocal. Variations may occur, such that splicing can become alternative, thereby participating in increasing protein variability and restricting the gap that exists between the relatively low number of genes - between 20,000 and 25,000 in humans - and the much higher number of distinct proteins - at least 100,000. In addition, although alternative pre-mRNA splicing often fulfils cell-specific needs, many aberrant splicing events can happen and lead to either hereditary or acquired diseases such as neurodegenerative diseases or cancers. In those cases, alternative splicing events may serve as disease-associated markers, or even as targets for corrective approaches. In this review, we will summarize the main aspects of regulated alternative splicing. We will present the spliceosome, a large ribonucleoprotein complex that orchestrates the splicing reactions and that was recently identified as a preferential target for mutations in several pathologies. We shall discuss some spliceosome-associated defects linked to either cis (i.e on the DNA) or trans (e.g. in proteins) alterations of splicing machinery, like those that have been reported in genetic or acquired diseases.