Mécanismes d'action et de résistance des Adénovirus C au Brincidofovir

Abstract : Human adenoviruses (HAdV) are responsible for significant morbidity and mortality in hematopoietic stem cell (HSC) recipients, particularly in children.Brincidofovir (BCV) prodrug of cidofovir is an analogue of cytosine. It inhibits the DNA polymerase (DNA pol) activity of HAdV by stopping elongation and competition with natural nucleotides.The objectives of this work are to study the genetic diversity of the pol DNA of HAdV and to set up tools for the analysis of HAdV C sensitivity and resistance to BCV.In a first part, we carried out the complete sequencing of the DNA pol gene of 60 clinical viral strains. The results show a nucleotide variability of 9.9% and peptide variability of 8.7%, variable according to the type. The most variable region is the NH2 terminal region with 44.2% of the amino acid mutations. None of the mutations described as associated with CDV or BCV resistance were detected. Polymorphism analysis has also shown that DNA pol sequences can identify sources of transmission during an outbreak. Finally, our observations suggest the low probability of recombination events as a factor of evolution of the gene coding for the DNA pol. In a second part, we developed a reverse genetic strategy from two plasmids, one coding for the whole genome deleted from the DNA pol (Adv-deltaPol) and one coding only for the viral DNA pol (Pol). 3 kilobase) in which the mutations are introduced by site-directed mutagenesis (Pol-mutated). After a bacterial recombination step into a single plasmid (Adv-deltaPol-Pol-mutated), 293T cells are transfected to produce the recombinant viruses. BCV susceptibility of mutated viruses will be analyzed on A549 cells. A total of 12 Pol-mutated plasmids were produced, recombinant virus L677F was obtained. Production of other recombinant viruses is ongoing.
Keywords : Brincidofovir
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Linda Feghoul. Mécanismes d'action et de résistance des Adénovirus C au Brincidofovir. Virologie. Université Sorbonne Paris Cité, 2017. Français. ⟨NNT : 2017USPCC303⟩. ⟨tel-02305971⟩

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