Periodic Reporting for period 1 - DELV (DELta Virus infection in animal and human hosts)
Período documentado: 2022-10-01 hasta 2025-03-31
The emergence and rapid transmission of viruses pose increasing risks and challenges to modern societies,
threatening public health and economic stability. A thorough understanding of basic virology is therefore
critical for an informed development of preventive and control strategies. Although for more than 40 years the
only known member of deltaviruses was the human Hepatitis Delta virus (HDV), it was recently discovered
that HDV-like agents are present in a variety of animal vectors and reservoirs including bats, rats, snakes, birds
and insects. Metagenomic data indicate that these satellite viruses possess an unrecognized host shifting
capacity enabling them to cross the species barrier. As no efficient antiviral treatment is available against HDV,
the emergence of novel deltaviruses poses a significant potential threat to human health. To date, animal
deltaviruses have not been functionally characterized and little is known about their basic biology. The
project (DELV) aims to generate essential knowledge about the biology of deltaviruses, their
interactions with host cells, their zoonotic potential and evolutionary fitness. Using newly generated deltavirus
molecular clones coupled to unbiased proteomic and genetic approaches, DELV aims to identify host factors
interacting with deltaviruses in their natural animal and human hosts. Further, it aims determine and
characterize host factors that are essential for deltavirus replication in the human host when they cross the
species barrier. Finally, DELV seeks to discover viral elements favoring deltavirus host shifting and
adaptation capacities. DELV aims to shape novel paradigms in virology, RNA biology and host pathogen
interactions. Knowledge generated through DELV will guide the development of novel antiviral
strategies and will have profound implications for understanding the ecology and evolution of these newly
discovered, yet mysterious, viral elements.
threatening public health and economic stability. A thorough understanding of basic virology is therefore
critical for an informed development of preventive and control strategies. Although for more than 40 years the
only known member of deltaviruses was the human Hepatitis Delta virus (HDV), it was recently discovered
that HDV-like agents are present in a variety of animal vectors and reservoirs including bats, rats, snakes, birds
and insects. Metagenomic data indicate that these satellite viruses possess an unrecognized host shifting
capacity enabling them to cross the species barrier. As no efficient antiviral treatment is available against HDV,
the emergence of novel deltaviruses poses a significant potential threat to human health. To date, animal
deltaviruses have not been functionally characterized and little is known about their basic biology. The
project (DELV) aims to generate essential knowledge about the biology of deltaviruses, their
interactions with host cells, their zoonotic potential and evolutionary fitness. Using newly generated deltavirus
molecular clones coupled to unbiased proteomic and genetic approaches, DELV aims to identify host factors
interacting with deltaviruses in their natural animal and human hosts. Further, it aims determine and
characterize host factors that are essential for deltavirus replication in the human host when they cross the
species barrier. Finally, DELV seeks to discover viral elements favoring deltavirus host shifting and
adaptation capacities. DELV aims to shape novel paradigms in virology, RNA biology and host pathogen
interactions. Knowledge generated through DELV will guide the development of novel antiviral
strategies and will have profound implications for understanding the ecology and evolution of these newly
discovered, yet mysterious, viral elements.
The DELV project aims at understanding the molecular basis of the deltavirus family (Kolmioviridae) interaction with animal and human host cells. Until few years ago, the only known representative of this viral family was Hepatitis D virus, a satellite of Hepatitis B virus that infects the human liver and causes serious liver disease (e.g. cirrhosis, hepatocellular carcinoma). Few years ago, several studies have discovered HDV-like sequences in a variety of animal species including birds, bats, rodents, snakes, frogs and insects. These newly discovered viral elements were never found with HBV or an HBV-like viruses and were not exclusively found in livers of infected animals but could be detected in other tissues including the gastrointestinal tract, lung and brain tissues. Before starting this project, very little was known about the lifecycle of these newly discovered animal viral satellites. For instance, are they able to replicate in any animal cell lines they access? Do they depend on the same factors as HDV to complete their lifecycle? What cellular factors can restrict their replication? What are the rules that govern their association with helper viruses? What is their evolutionary trajectory once the cross the species barrier infecting human cells?
During this first reporting period the work that was performed has helped confirm several working hypotheses as well as answering several questions that were initially asked. We have now established several read-outs to detect and follow deltavirus infection in different cell lines and have established an in vivo mouse model. Indeed, a study from our lab funded by the DELV project has been published in the journal PLoS Pathogens (DOI: 10.1371/journal.ppat.1012060) earlier this year. In this study we establish several tools essential to study animal deltaviruses: 1- we developed single molecule RNA Fluorescent In Situ Hybridization (smFISH) coupled to immunofluorescence (IF) and confocal microscopy to visualize viral RNAs and proteins respectively, 2- we established viral pseudo-typing with heterologous envelope proteins (e.g. Vesicular Stomatitis Virus glycoprotein or VSV-G) , 3- we developed Northern and Western blot assays to quantify both viral RNA and protein production and 4- we took advantage of the hydrodynamic tail-vein injection method to develop a mouse model recapitulating deltavirus replication in the livers of injected animals. Most importantly, by comparing replication of three deltaviruses: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo our study shows that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis thus layed the foundation for the discovery of specific host factors controlling deltavirus host-shifting.
Most significant achievements
- Publication of the first research article by the team in 2024 published in PLoS Pathogens, Khalfi et al, Comparative analysis of human, rodent and snake deltavirus replication. 2024 Mar 5;20(3):e1012060. doi: 10.1371/journal.ppat.1012060.
- Publication of a review article in 2023 in the journal Antiviral Research, Khalfi et al, Hepatitis D virus: Improving virological knowledge to develop new treatments. Antiviral Res. 2023 Jan;209:105461. doi: 10.1016/j.antiviral.2022.105461. Epub 2022 Nov 14. PMID: 36396025.
- Publication of a review article in 2024 in the journal Trends in Microbiology, Gerber-Tichet et al, Toll-like receptor 4 - a multifunctional virus recognition receptor. Trends Microbiol. 2024 Aug 22:S0966-842X(24)00171-9. doi: 10.1016/j.tim.2024.07.001. Epub ahead of print. PMID: 39179422.
- Best poster prize for the post-doctoral fellow Pierre Khalfi in the 2023 international HBV meeting held in Kobe, Japan.
- Best presentation prize for the PhD student Zoé Denis in the HepDART 2023 conference held in Los Cabos, Mexico.
During this first reporting period the work that was performed has helped confirm several working hypotheses as well as answering several questions that were initially asked. We have now established several read-outs to detect and follow deltavirus infection in different cell lines and have established an in vivo mouse model. Indeed, a study from our lab funded by the DELV project has been published in the journal PLoS Pathogens (DOI: 10.1371/journal.ppat.1012060) earlier this year. In this study we establish several tools essential to study animal deltaviruses: 1- we developed single molecule RNA Fluorescent In Situ Hybridization (smFISH) coupled to immunofluorescence (IF) and confocal microscopy to visualize viral RNAs and proteins respectively, 2- we established viral pseudo-typing with heterologous envelope proteins (e.g. Vesicular Stomatitis Virus glycoprotein or VSV-G) , 3- we developed Northern and Western blot assays to quantify both viral RNA and protein production and 4- we took advantage of the hydrodynamic tail-vein injection method to develop a mouse model recapitulating deltavirus replication in the livers of injected animals. Most importantly, by comparing replication of three deltaviruses: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo our study shows that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis thus layed the foundation for the discovery of specific host factors controlling deltavirus host-shifting.
Most significant achievements
- Publication of the first research article by the team in 2024 published in PLoS Pathogens, Khalfi et al, Comparative analysis of human, rodent and snake deltavirus replication. 2024 Mar 5;20(3):e1012060. doi: 10.1371/journal.ppat.1012060.
- Publication of a review article in 2023 in the journal Antiviral Research, Khalfi et al, Hepatitis D virus: Improving virological knowledge to develop new treatments. Antiviral Res. 2023 Jan;209:105461. doi: 10.1016/j.antiviral.2022.105461. Epub 2022 Nov 14. PMID: 36396025.
- Publication of a review article in 2024 in the journal Trends in Microbiology, Gerber-Tichet et al, Toll-like receptor 4 - a multifunctional virus recognition receptor. Trends Microbiol. 2024 Aug 22:S0966-842X(24)00171-9. doi: 10.1016/j.tim.2024.07.001. Epub ahead of print. PMID: 39179422.
- Best poster prize for the post-doctoral fellow Pierre Khalfi in the 2023 international HBV meeting held in Kobe, Japan.
- Best presentation prize for the PhD student Zoé Denis in the HepDART 2023 conference held in Los Cabos, Mexico.
We observe that deltaviruses form phase-separated membraneless viral RNA-protein condensates in nuclei of persistently infected cells.