Nouvelles approches thérapeutiques pour contrer les infections causées par le metapneumovirus humain

Les infections respiratoires sont une cause importante de morbidité et de mortalité à travers le monde. D’ailleurs, de nouveaux virus respiratoires sont continuellement identifiés, dont le metapneumovirus humain (hMPV) en 2001. Ce virus est maintenant reconnu comme une cause fréquente d’infections respiratoires sévères chez les jeunes enfants, telles que des pneumonies et des bronchiolites. Il se classe d’ailleurs en 2e ou 3e position après le virus respiratoire syncytial, en alternance avec les virus influenza. La majorité des infections sévères causées par le hMPV touche les individus dont le système immunitaire est plus faible c’est-à-dire les jeunes enfants, les personnes âgées et les sujets immunosupprimés. Diverses études démontrent qu’une infection respiratoire virale sévère en bas âge peut prédisposer un enfant à développer de l’asthme durant sa vie. Malgré cela, les médecins n’ont toujours aucun vaccin ni traitement à leur disposition. L’analyse du génome et de la fonction des protéines du hMPV nous offre maintenant des pistes prometteuses pour le développement d’antiviraux. De plus, un modèle murin bien établi permet désormais l’étude in vivo de ces antiviraux. Ce projet de doctorat avait donc comme objectif de développer des approches thérapeutiques pour contrer les infections causées par le hMPV. Pour ce faire, deux étapes de l’infection ont été ciblées : l’entrée du virus dans la cellule hôte par fusion, et la réplication du génome viral à l’intérieur de la cellule infectée. La première molécule identifiée avec succès est un inhibiteur de fusion, le peptide HRA2, qui s’est révélé hautement efficace in vitro et in vivo contre le hMPV. Le deuxième groupe de molécules est formé de petits ARN interférants (siRNAs) dirigés contre des gènes essentiels à la réplication du hMPV. L’interférence à l’ARN est d’ailleurs une discipline en plein essor depuis sa récente identification. Deux siRNAs ciblant la nucléoprotéine et la phosphoprotéine du hMPV se sont avérés extrêmement actifs contre ce virus in vitro et des résultats très encourageants ont été obtenus chez la souris.Respiratory infections are a leading cause of morbidity and mortality worldwide. New viruses are constantly discovered and one of them, the human Metapneumovirus (hMPV), was identified in 2001. hMPV causes upper and lower respiratory infections ranging from cold- or flu-like illnesses to more severe and life-threatening infections such as pneumonia and bronchiolitis. hMPV is only second or third to human respiratory syncytial virus in frequency and sometimes as frequent as human influenza viruses in causing respiratory infections. The majority of severe infections caused by hMPV are seen in individuals having the weakest immune system such as young children, elderly and immunocompromised subjects. Many studies tend to demonstrate that severe viral respiratory infection during childhood can lead to asthma development later in life. Unfortunately, physicians have no vaccine or treatment to fight against severe hMPV infections. Genome and protein fonction analyses are offering promising avenues for the development of effective antivirals. Moreover, hMPV pathogenesis has been described in a murine model which now offers a convenient way to evaluate candidate molecules. This PhD project main goal was therefore to design new molecules against hMPV infections. For that matter, two steps of hMPV replication cycle were targeted, i.e., virus entry into the host cell which happens by a fusion mechanism, and viral genome replication inside the infected cell. The first molecule successfully identified is a fusion inhibitor, the HRA2 peptide that showed highly potent activity against hMPV in vitro but also in vivo. The second group of molecules corresponds to small interfering RNAs (siRNAs) targeting genes essential for hMPV replication. RNA interference has been a rapidly growing field since its first description in the 1990’s. Here, we identified two highly effective siRNAs against hMPV in vitro which target the nucleoprotein and phosphoprotein. Evaluation of these siRNAs in a murine model of hMPV infection also showed great possibilities

Promotion of Hendra virus replication by microRNA 146a

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease

Analysis of Replication Kinetics of the Human Metapneumovirus in Different Cell Lines by Real-Time PCR

Human metapneumovirus (hMPV) is associated with acute respiratory tract disease especially in young children. Using a quantitative real-time TaqMan PCR, we analyzed the replication kinetics of hMPV in different cell lines. Our results indicate that hMPV replicates slightly more efficiently in LLC-MK2 than in Vero cells and poorly in HEp-2 cells

A Functional Genomics Approach to Henipavirus Research: The Role of Nuclear Proteins, MicroRNAs and Immune Regulators in Infection and Disease.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are zoonotic RNA viruses that cause lethal disease in humans and are designated as Biosafety Level 4 (BSL4) agents. Moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. Due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. In this chapter we describe recent loss-of-function (i.e. RNAi) functional genomics screens that shed light on the henipavirus-host interface at a genome-wide level. Further to this, we cross-reference RNAi results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. These functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. Furthermore these studies will underpin future efforts to define the role of nucleolar host-virus interactions in infection and disease

IFN regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the CNS.

IFN regulatory factor (IRF) 8 is a transcription factor that has a key role in the cellular response to IFN-γ and is pivotal in myeloid cell differentiation. Whether IRF8 plays a role in the development and function of microglia, the tissue-resident myeloid cells of the brain, is unknown. Here, we show IRF8 is a constitutively produced nuclear factor in microglia, which suggested that IRF8 might also be a key homeostatic transcriptional determinant of the microglial cell phenotype. In support of this, in mice with a targeted disruption of the IRF8 gene, microglia were increased in number and showed gross alterations in morphology and surface area. In situ analysis of some key myeloid markers revealed that IRF8-deficient microglia had significantly reduced levels of Iba1, but increased levels of CD206 (mannose receptor) and F4/80 as well as increased tomato lectin binding. Analysis of microglia ex vivo revealed IRF8-deficient microglia had significantly increased levels of CD45, CD11b and F4/80, but significantly decreased levels of the chemokine receptors CCR2, CCR5 and CX3CR1. The known involvement of some of these molecular markers in membrane dynamics and phagocytosis led us to examine the phagocytic capacity of cultured IRF8-deficient microglia, however, this was found to be similar to wild type microglia. We conclude IRF8 is a constitutively produced nuclear factor in resident microglia of the CNS being a crucial transcriptional determinant of the phenotype of these cells in the healthy brain