Therapeutic targets for human adenovirus infections

Les adénovirus humains (HAdV) sont des virus à ADN responsables d’une importante morbi-mortalité chez les patients allogreffés de cellules souches hématopoïétiques (CSH). L’étude et le traitement des infections à HAdV représentent donc des enjeux majeurs. La diversité génétique et les facteurs de virulence des principaux types associés aux greffes de CSH restent cependant peu connus et les stratégies antivirales sont très peu développées. Il n’existe actuellement aucun traitement approuvé contre les infections à HAdV. Dans une première partie nous avons étudié la diversité génétique de génomes complets de HAdV de l’espèce C isolés chez des enfants greffés de CSH et non greffés. Cette étude a permis de caractériser un nouveau type de HADV-C et de mettre en évidence l’existence de nombreux événements de recombinaison au niveau de la région précoce de leurs génomes, pouvant impacter la virulence des HAdV. Le brincidofovir (BCV) est un inhibiteur d’ADN polymérase viral en développement. Des mutations de résistance au niveau de l’ADN polymérase virale ont été décrites in vitro. Dans la deuxième partie de ce travail, nous avons développé un modèle de HAdV recombinant permettant de déterminer l’impact de ces mutations sur les capacités réplicatives du virus et sur sa sensibilité aux inhibiteurs d’ADN polymérase. Des données suggèrent aussi l’existence d’une activité antivirale du BCV indépendante de l’ADN polymérase. Dans la troisième partie de ce travail nous avons exploré l’impact du BCV sur le transcriptome de la cellule. Nous avons pu mettre en évidence une action du BCV sur l’expression cellulaire de voies de signalisation participant au contrôle de la réplication virale. Enfin, nous avons recherché de nouvelles molécules anti-HAdV en utilisant différentes stratégies. Nous avons pu identifier 12 molécules possédant une activité anti-HADV in vitro. L’ensemble de nos travaux ont permis d’approfondir les connaissances sur la diversité des HAdV et sur le mécanisme d’action des antiviraux utilisés dans ces infections. Nous avons également mis en lumière plusieurs cibles thérapeutiques cellulaires potentielles.Human adenoviruses (HAdV) are DNA viruses responsible for a significant morbidity and mortality in children with allogeneic hematopoietic stem cell transplant (HSCT). The study and treatment of HAdV infections are therefore a major challenge. However, the genetic diversity and virulence factors of the main types associated with HSCT recipients remain poorly understood and there are few antiviral strategies. There is currently no approved treatment for HAdV infections. In a first part, we studied the genetic diversity of complete HAdV-C species genomes isolated from children with and without HSCT. This study enabled to characterize a new type of HAdV-C and to highlight the existence of several recombination events in the region coding for early genes that might impact HAdV virulence. Brincidofovir is a viral DNA polymerase inhibitor. Resistance mutations in viral DNA polymerase have been described in vitro. In the second part of this work, we developed a recombinant HAdV model to determine the impact of these mutations on the virus' replication capacity and its susceptibility to DNA polymerase inhibitors. Furthermore, data suggest the existence of DNA polymerase-independent antiviral activity of BCV. In the third part of this work we explored the impact of BCV on the transcriptome of the cell. We were able to demonstrate an action of BCV on the cellular expression of signalling pathways involved in the control of viral replication. Finally, we investigated new anti-HAdV molecules using different strategies. We were able to identify twelve molecules with anti-HAdV activity in vitro. In conclusion, this work has clarified our knowledge of HAdV diversity and the mechanism of action of the antivirals used in HAdV infections. We have also highlighted several potential cellular therapeutic targets

Laboratory-based strategy using a new marketed polymerase chain reaction assay to manage diarrheic episodes among patients from rehabilitation and long-term care facilities

Management of Norovirus and Clostridium difficile gastroenteritis is challenging for rehabilitation and long-term care facilities. We evaluated the contribution of a 2-step laboratory-based strategy, including a new ready-to-use Norovirus polymerase chain reaction assay to promote isolation precautions. C difficile and Norovirus were successively identified from 17% and 23% of 52 episodes of diarrhea, respectively, during the winter season, leading to 100% adequate isolation measures. In patient populations with numerous risk factors for diarrhea, a combined laboratory-based approach could improve infection control

Impact of the COVID-19 pandemic on antiviral drug development for other community-acquired respiratory viruses’ infections

The coronavirus disease 2019 (COVID-19) pandemic indirectly resulted in missed therapeutic opportunities for many diseases. Here we focus on community-acquired respiratory viruses other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) [respiratory syncytial virus, parainfluenza and influenza A], and highlight the pandemics impact on clinical trials to develop novel therapies for other severe respiratory viral infections. We retrospectively reviewed inclusion rates within respiratory antiviral clinical trials in comparison with all other clinical trials in our clinical investigations center, before and during the COVID-19 pandemic. As opposed to the remaining clinical trials developed within our unit, respiratory antiviral trials inclusion rates did not recover after the initial recruitment decrease observed across all trials during the first pandemic wave. These results were discussed in the context of non-COVID-19 respiratory viral infection rates within our center, showing a general decline in seasonal respiratory viruses spread since the COVID-19 pandemic onset. Virus epidemiology changes upon the wide SARS-CoV-2 expansion as well as the lifestyle changes globally adopted to prevent SARS-CoV-2 transmission could have therefore contributed to the negative impact of the COVID-19 pandemic on antiviral drug development. Our study highlights the peculiarity of respiratory antiviral drug development during the COVID-19 pandemic era and describes potential explanations for such drug development halting

Genetic diversity of the human adenovirus species C DNA polymerase

International audienceBackground: Human Adenovirus (HAdV) are responsible for severe infections in hematopoietic stem cells transplant (HSCT) recipient, species C viruses being the most commonly observed in this population. There is no approved antiviral treatment yet. Cidofovir (CDV), a cytidine analog, is the most frequently used and its lipophilic conjugate, brincidofovir (BCV), is under clinical development. These drugs target the viral DNA polymerase (DNA pol). Little is known about the natural polymorphism of HAdV DNA pol in clinical strains. Methods: We assessed the inter- and intra-species variability of the whole gene coding for HAdV DNA pol of HAdV clinical strains of species C. The study included 60 species C HAdV (21 C1, 27 C2 and 12 C5) strains isolated from patients with symptomatic infections who had never experienced CDV or BCV treatments and 20 reference strains. We also evaluated the emergence of mutations in thrirteen patients with persistent HAdV infection despite antiviral treatment. Results: We identified 356 polymorphic nucleotide positions (9.9% of the whole gene), including 102 positions with nonsynonymous mutations (28.0%) representing 8.7% of all amino acids. The mean numbers of nucleotide and amino acid mutations per strain were 23.1 (+/- 6.2) and 5.2 (+/- 2.4) respectively. Most of amino acid substitutions (60.6%) were observed in one instance only. A minority (13.8%) were observed in more than 10% of all strains. The most variable region was the NH2 terminal domain (44.2% of amino acid mutations). Mutations in the exonuclease domain accounted for 27.8%. The binding domains for the terminal protein (TPR), TPR1 and TPR2, presented a limited number of mutations, which were nonetheless frequently observed (62.5% and 58.8% of strains for TPR1 and TPR2, respectively). None of the mutations associated with CDV or BCV resistance were detected. In patients receieving antiviral drugs with persistent HAdV replication, we identified a new mutation in the NH2 terminal region. Conclusions: Our study shows a high diversity in HAdV DNA pol sequences in clinical species C HAdV and provides a comprehensive mapping of its natural polymorphism. These data will contribute to the interpretation of HAdV DNA pol mutations selected in patients receiving antiviral treatments

DNA ultra-sensitive quantification, a technology for studying HIV unintegrated linear DNA

International audienceUnintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using molecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11 days in resting CD4+ T cells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ in vivo to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules

Genotypic and Phenotypic Diversity of the Replication-Competent HIV Reservoir in Treated Patients

International audienceIn HIV infection, viral rebound after treatment discontinuation is considered to originate predominantly from viral genomes integrated in resting CD4+ T lymphocytes. Replication-competent proviral genomes represent a minority of the total HIV DNA. While the quantification of the HIV reservoir has been extensively studied, the diversity of genomes that compose the reservoir was less explored. Here, we measured the genotypic and phenotypic diversity in eight patients with different treatment histories. Between 4 and 14 (mean, 8) individual viral isolates per patient were obtained using a virus outgrowth assay, and their near-full-length genomes were sequenced. The mean pairwise distance (MPD) observed in different patients correlated with the time before undetectable viremia was achieved (r = 0.864, P = 0.0194), suggesting that the complexity of the replication-competent reservoir mirrors that present at treatment initiation. No correlation was instead observed between MPD and the duration of successful treatment (mean, 8 years; range, 2 to 21 years). For 5 of the 8 patients, genotypically identical viral isolates were observed in independent wells, suggesting clonal expansion of infected cells. Identical viruses represented between 25 and 60% of the isolates (mean, 48%). The proportion of identical viral isolates correlated with the duration of treatment (r = 0.822, P = 0.0190), suggesting progressive clonal expansion of infected cells during ART. A broader range of infectivity was also observed among isolates from patients with delayed viremia control (r = 0.79, P = 0.025). This work unveiled differences in the genotypic and phenotypic features of the replication-competent reservoir from treated patients and suggests that delaying treatment results in increased diversity of the reservoir. IMPORTANCE In HIV-infected and effectively treated individuals, integrated proviral genomes may persist for decades. The vast majority of the genomes, however, are defective, and only the replication-competent fraction represents a threat of viral reemergence. The quantification of the reservoir has been thoroughly explored, while the diversity of the genomes has been insufficiently studied. Its characterization, however, is relevant for the design of strategies aiming the reduction of the reservoir. Here, we explored the replication-competent near-full-length HIV genomes of eight patients who experienced differences in the delay before viremia control and in treatment duration. We found that delayed effective treatment was associated with increased genetic diversity of the reservoir. The duration of treatment did not impact the diversity but was associated with higher frequency of clonally expanded sequences. Thus, early treatment initiation has the double advantage of reducing both the size and the diversity of the reservoir

Severe Pneumonia Associated with Adenovirus Type 55 Infection, France, 2014

International audienceHuman adenoviruses (HAdVs) comprise 70 recognized genotypes (as of February 15, 2016; http://hadvwg.gmu.edu/) and are frequently associated with mild and acute upper respiratory tract infections, depending on virus type and host immune status (1). HAdV type 55 (HAdV-55) has recently reemerged as a highly virulent pathogen, causing severe and sometimes fatal pneumonia among immunocompetent adults, particularly in Asia (2–4). Formerly known as HAdV-11a, HAdV-55 is a genotype resulting from recombination between HAdV-11 and HAdV-14 (5). We report 2 cases of severe pneumonia associated with HAdV-55 infection in Franc

A multi-disciplinary comparison of great ape gut microbiota in a central African forest and European zoo

We are grateful to the inhabitants and authorities in southeastern Cameroon for their warm welcome and their support of this study. We also acknowledge the invaluable assistance of the European zoo and its employees where we conducted the investigation. We also greatly appreciate the contributions of Olivia Cheny of the Center for Translational Science at the Institut Pasteur.International audienceComparisons of mammalian gut microbiota across different environmental conditions shed light on the diversity and composition of gut bacteriome and suggest consequences for human and animal health. Gut bacteriome comparisons across different environments diverge in their results, showing no generalizable patterns linking habitat and dietary degradation with bacterial diversity. The challenge in drawing general conclusions from such studies lies in the broad terms describing diverse habitats ("wild", "captive", "pristine"). We conducted 16S ribosomal RNA gene sequencing to characterize intestinal microbiota of free-ranging sympatric chimpanzees and gorillas in southeastern Cameroon and sympatric chimpanzees and gorillas in a European zoo. We conducted participant-observation and semi-structured interviews among people living near these great apes to understand better their feeding habits and habitats. Unexpectedly, bacterial diversity (ASV, Faith PD and Shannon) was higher among zoo gorillas than among those in the Cameroonian forest, but zoo and Cameroonian chimpanzees showed no difference. Phylogeny was a strong driver of species-specific microbial composition. Surprisingly, zoo gorilla microbiota more closely resembled that of zoo chimpanzees than of Cameroonian gorillas. Zoo living conditions and dietary similarities may explain these results. We encourage multidisciplinary approach integrating environmental sampling and anthropological evaluation to characterize better diverse environmental conditions of such investigations. Over the last decade, numerous studies have demonstrated the importance of environmental changes on the mammalian gut microbiome, which is strongly associated with host metabolic, immune, and neurological functions 1. Broad-ranging influences, including host genetics, living conditions, diet, stress, and antibiotic use can affect gut microbial diversity 2-5. Among these influences, diet and living conditions have been evaluated for human and animal populations, entailing significant effects on gut microbiota and consequences for human and animal health. Adverse microbial profile shifts, for instance, have been associated with dysbiosis and wide-ranging diseases among human beings, from obesity to pediatric environmental enteropathy, and from autism to asthma 2,6,7. Outside of laboratory conditions, disentangling the effects of living and dietary conditions on gut microbial composition from other influences remains a complex question. In humans, such questions have catalyzed multiple studies comparing environmental and gut microbiota between "westernized" and "rural" peoples 8-10. Among other mammalian populations, degradation in habitat quality affects the diversity of available flora and fauna for consumption, and in some cases, is associated with declines in microbial gut composition 11-14. Microbiome OPE