USE OF FXR AGONISTS FOR TREATING AN INFECTION BY HEPATITIS D VIRUS

Abstract

The present invention relates to the use of a farnesoid X receptor (FXR) agonist for the treatment of hepatitis D infection.

Claims

1-15. (canceled)

16. A method for the treatment of Hepatitis D virus (HDV) infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a farnesoid X receptor (FXR) agonist.

17. The method of claim 16, wherein the subject suffers from a chronic HDV infection.

18. The method of claim 16, wherein the FXR agonist is a selective FXR agonist.

19. The method of claim 16, wherein the FXR agonist is selected from the group consisting of LJN452 (Tropifexor), LMB763 (Nidufexor), GS-9674 (Cilofexor), PX-102 (PX-20606), PX-104 (Phenex 104), OCA (Ocaliva), EDP-305, TERN-101 (LY2562175), MET-409, GW4064, WAY362450 (Turofexorate isopropyl), Fexaramine, AGN242266 (AKN-083), BAR502, and EYP001.

20. The method of claim 16, wherein the FXR agonist is EYP001.

21. The method of claim 16, wherein said FXR agonist is administered in combination with an interferon alpha (IFN-α), an interferon lambda or a pegylated form thereof.

22. The method of claim 16, wherein said FXR agonist is administered in combination with an anti-HDV agent.

23. The method of claim 22, wherein said anti-HDV agent is selected from the group consisting of ribavirin, ritonavir, lonafarnib and EBP 921.

24. The method of claim 16, wherein said FXR agonist is administered in combination with an anti-HBV agent.

25. The method of claim 24, wherein said anti-HBV agent is selected from the group consisting of lamivudine, adefovir, telbivudine, entecavir, tenofovir and emtricitabine.

26. The method of claim 16, wherein said FXR agonist is administered in combination with an anti-HBV/HDV agent

27. The method of claim 26, wherein said anti-HBV/HDV agent is selected from the group consisting of ezetimibe, myrcludex B, nucleic acid polymer REP 2139 and nucleic acid polymer REP 2165.

28. The method of claim 16, wherein the subject has failed to respond to a previous treatment for HDV infection.

29. The method of claim 28, wherein the previous treatment is a treatment with PEG-IFNα.

30. The method of claim 28, wherein the previous treatment is a treatment with an anti-HDV agent.

31. The method of claim 26, wherein said anti-HBV/HDV agent is a nucleoside analog, a nucleic acid polymer or a sodium taurocholate cotransporting polypeptide (NTCP) inhibitor.

32. The method of claim 24, wherein said anti-HBV agent is a nucleoside analog.

33. The method of claim 22, wherein said anti-HDV agent is a nucleoside analog or a farnesyl transferase inhibitor.

34. The method of claim 21, wherein said interferon alpha (IFN-α), interferon lambda or a pegylated form thereof is selected from the group consisting of IFN-α1a, IFN-α1b, IFN-α2a, IFN-α2b, and IFN-λ1a or a pegylated form thereof.

Description

FIGURES

[0136] FIG. 1. FXRα agonist inhibits HDV replication in HBV-HDV coinfected dHepaRG cells. Differentiated HepaRG cells were infected with HBV at a MOI of 100 GE/cell and with HDV at a MOI of 10 GE/cell. From day 3 to 13 post-infection, cells were treated with 10 μM of GW4064, interferon α-2a (1000 IU/mL) or vehicle. Cells and supernatants were harvested at day 13 for intracellular HBV and HDV RNA and secreted antigens quantification. Results are the mean+/−SD of two experiments performed with three biological replicates.

[0137] FIG. 2. FXRα agonist inhibits HDV replication in HBV-infected dHepaRG super-infected with HDV. Differentiated HepaRG cells were infected with HBV at a MOI of 100 GE/cell and 7 days later with HDV at a MOI of 10 GE/cell. From day 10 to day 17 post-HBV infection, cells were treated with 1, 5 or 10 μM of GW4064, interferon α-2a (1000 IU/mL) or vehicle. Cells and supernatants were harvested at day 17 for intracellular HDV RNA quantification. Results are the mean+/−SD of three experiments (in dHepaRG) performed with three biological replicates.

[0138] FIG. 3. FXRα agonist inhibits HDV replication in HBV-infected PHH super-infected with HDV. PHH were infected with HBV at a MOI of 100 GE/cell and 4 days later with HDV at a MOI of 10 GE/cell. From day 7 to day 14 post-HBV infection, cells were treated with 1, 5 or 10 μM of GW4064, interferon α-2a (1000 IU/mL) or vehicle. Cells and supernatants were harvested at day 14 post HBV infection for intracellular HDV RNA quantification. Results are the mean+/−SD of one experiment performed with three biological replicates.

[0139] FIG. 4. FXRα agonist inhibits the production of HDV proteins in HBV-infected dHepaRG superinfected with HDV. Differentiated HepaRG cells were infected with HBV at a MOI of 100 GE/cell and 7 days later with HDV at a MOI of 10 GE/cell. From day 10 to day 17 post-HBV infection, cells were treated with 1, 5 or 10 μM of GW4064, interferon α-2a (1000 IU/mL) or vehicle. Cells were harvested at day 17 post HBV infection and lysed for protein extraction and WB analyses. Graphs represent the densitometry analyses of the respective blots and results are presented as ratios of HDAgs normalized to the levels of B-tubulin.

[0140] FIG. 5. FXRα agonist inhibits the production of HDV proteins in HBV-infected PHH superinfected with HDV. PHH were infected with HBV at a MOI of 100 GE/cell and 4 days later with HDV at a MOI of 10 GE/cell. From day 7 to day 14 post-HBV infection, cells were treated with 1, 5 or 10 μM of GW4064, interferon α-2a (1000 IU/mL) or vehicle. Cells and supernatants were harvested at day 14 post HBV infection for intracellular HDV RNA quantification and lysed for protein extraction and WB analyses. Graphs represent the densitometry analyses of the respective blots and results are presented as ratios of HDAgs normalized to the levels of B-tubulin.

[0141] FIG. 6. FXRα agonists inhibit HDV replication in monoinfected dHepaRG cells. Differentiated HepaRG cells were infected with HDV at a MOI of 25 GE/cell. From day 4 to day 11 post-infection, cells were treated with 1 or 10 μM of GW4064, 10 μM of 6-ECDCA and 1 μM of tropifexor. At day 11 post HDV infection, cells were collected and total intracellular HDV RNAs were quantified by qPCR.

[0142] FIG. 7. FXRα agonists decrease the amount of HDV genomic RNA in monoinfected dHepaRG cells. Differentiated HepaRG cells were infected with HDV at a MOI of 25 GE/cell. From day 4 to day 11 post-infection, cells were treated with 1 or 10 μM of GW4064, 10 μM of 6-ECDCA and 1 μM of tropifexor. At day 11 post HDV infection, cells were collected and HDV genomic RNA was analyzed by Northern Blot.

[0143] FIG. 8. Decrease of the levels of nascent HDV RNAs in HBV/HDV co-infected dHepaRG treated with FXRα agonists. dHepaRG were co-infected with HBV (100 vge/cell) and with HDV (10 vge/cell). Six days later, cells were treated with GW4064 (10 μM) or with interferon α-2a (1000 U/mL) for 4 days. Cells were incubated with labelled uridin or not (mock-EU) for 2 h, washed and harvested. Total intracellular HDV RNAs as well as EU-labelled HDV RNAs (nascent intracellular HDV RNAs) were isolated and quantified by RT-qPCR analyses. As a control, cells were treated with actinomycin D (10 μg/mL, ActD) 20 min before incubation with labelled uridin in order to block transcription of nascent RNAs. Results are the mean+/−SD one experiment performed with three biological replicates.

[0144] FIG. 9. GW4064 reduces the infectivity of HDV particles. dHepaRG cells were co-infected with HBV and HDV with 500 vge/cell for HBV and 50 vge/cell for HDV. Cells were treated or not 3 days later with GW4064 (10 μM), IFN-α (500 UI/mL) or lamivudine (LAM, 10 μM) for 10 days. (A) Supernatants of infected dHepaRG cells were collected, concentrated by PEG precipitation and the levels of extracellular HDV RNAs were assessed by qRT-PCR analyses. (B-C) Naïve HuH7.5-.sup.NTCP cells were infected with the different concentrated supernatants with (B) 500 vge/cell or (C) as indicated. Six days later, levels of intracellular HDV RNAs were assessed by RT-qPCR analyses. Results of RT-qPCR are the mean+/−SD of three independent experiments each performed with three biological replicates.

EXAMPLES

Results

[0145] To determine the impact of FXR agonists on HDV infection, in vitro infections were performed in differentiated HepaRG cells (dHepaRG) and primary human hepatocytes (PHH).

[0146] After differentiation, HepaRG cells are susceptible to infection with HDV virions produced in vitro, either in monoinfection, or coinfection and superinfection with HBV. In the case of HBV/HDV coinfected or superinfected cells, this model allows the study of all steps of the HDV replication cycle, including penetration into the cell, translocation of the viral genome into the nucleus, replication of the viral genome and synthesis of viral mRNAs, as well as later stages of the viral cycle with assembly and secretion of infectious virions bearing HBV HBs envelope proteins. In HDV monoinfected cells, all steps of the viral cycle can be explored except the assembly process as newly-synthesized HBV envelope proteins are lacking.

[0147] PHH are also susceptible to infection with HDV virions produced in vitro, either in monoinfection, or coinfection and superinfection with HBV.

Treatment with FXR Modulators Inhibit HDV Replication in HBV/HDV Coinfected HepaRG Cells.

[0148] The inventors first evaluated the impact of FXRα agonists on HDV replication in in vitro coinfected dHepaRG cells. Cells were simultaneously infected with HBV and HDV. Three days post-infection, cells were treated for 10 days with FXR agonist GW4064 at 10 μM or 1000 IU/mL of interferon α-2a. At day 13 post-infection, cells and supernatants were collected. The intracellular amounts of HDV and HBV RNAs were quantified as well as secreted HBe and HBs antigens.

[0149] The amount of total intracellular HDV RNA was decreased by GW4064 by 60% in HepaRG at 10 μM (FIG. 1A). This decrease of viral RNA was comparable to that observed with interferon α-2a. The anti-HBV activity of GW4064, which was previously described, was verified on the amount of intracellular HBV RNAs and secreted HBs and HBe antigens (FIGS. 1B, 1C and 1D).

Treatment with FXR Modulators Inhibit HDV Replication in HBV and HDV Superinfected Cells.

[0150] The impact of FXRα agonists on HDV replication was also evaluated in in vitro models of HDV superinfection of HBV-infected hepatocytes (both dHepaRG cells and PHH). Cells were successively infected with HBV and 7 days later with HDV. Three days post HDV infection, cells were treated for 7 days with GW4064 at 1, 5 and 10 μM or 1000 UI/mL interferon α-2a. The intracellular amount of total HDV RNA was quantified by RT-qPCR.

[0151] In both dHepaRG cells and PHH, treatment with FXR agonist GW4064 decreased the amount of total intracellular HDV RNA, up to 60% in HepaRG cells (FIG. 2) and 45% in PHH (FIG. 3) at 10 μM. The effect was already very pronounced at 1 μM. This decrease of viral RNA was comparable to that observed with 1000 IU/mL of interferon α-2a.

[0152] Importantly, treatment with GW4064 also decreased the amount of HDV antigens (HDAg) in both superinfected dHepaRG cells (FIG. 4) and PHH (FIG. 5), as detected by Western Blot analysis. Of note, FXRα agonist decreased both HDAg-L (large HDV antigen) and HDAg-S (small HDV antigen) in the same proportions, i.e. 75% reduction of their amount in both models. Inhibition of HDAgs was slightly higher following treatment with 10 μM of GW4064 than that obtained with 1000 IU/mL of interferon α-2a. Collectively these results indicate that FXRα agonist GW4064 likely inhibits HDV at the mRNA level, leading to a very strong inhibition at protein levels.

Treatment with FXR Modulators Inhibit HDV Replication in HDV Monoinfected Cells.

[0153] To determine if FXRα-mediated inhibition of HDV was independent of HBV, the inventors analyzed the impact of FXRα agonists in HDV mono-infected dHepaRG cells. Four days post-infection with HDV alone, cells were treated for seven days with 3 different FXRα agonists: 6-ECDCA at 10 μM, GW4064 at 1 and 10 μM and tropifexor at 1 μM. The impact of treatment on the amount of total HDV RNAs was analysed by RT-qPCR. The specific impact of FXR agonists on the amount of genomic HDV RNA was evaluated by Northern Blot analysis.

[0154] GW4064 at 10 μM, 6-ECDCA and tropifexor all decreased the amount of total HDV RNA by around 60%, as measured by RT-qPCR (FIG. 6) and also of genomic RNA detected by Northern Blot (FIG. 7).

[0155] Altogether these results indicate that FXRα agonists inhibit HDV infection in a way that is independent of their inhibitory effect on HBV infection. Moreover 3 different FXRα agonists with distinct structures showed comparable efficiency in HDV inhibition.

Treatment with FXR Modulators Inhibit Synthesis of Nascent HDV RNAs.

[0156] To get initial insight on the mode of action of FWR agonists on HDV, the inventors performed Run-ON assay in HBV/HDV coinfected dHepaRG cells to determine whether nascent HDV RNA could be also inhibited as total HDV RNA amount was shown to be. dHepaRG cells were simultaneously infected with HBV and HDV. Six days post-infection, cells were treated with 10 μM of GW4064 for 4 days before Run-On experiment. Results showed that GW4064 at 10 μM was indeed able to inhibit the synthesis of HDV RNA within 2 hours of staining with labeled uridin, thus suggesting that the initiation of HDV mRNA and or elongation could be impacted (FIG. 8).

Treatment with FXR Modulators Inhibit Specific Infectivity of Secreted HDV Viral Particles.

[0157] The impact of FXRα agonists on secretion and specific infectivity of HDV viral particles was evaluated in in vitro coinfected dHepaRG cells. Three days post-infection, cells were treated for 10 days with FXR agonist GW4064 at 10 μM or 1000 IU/mL of interferon α-2a or lamivudine at 10 μM. Supernatants were collected and concentrated using 8% PEG 8000. First, the secreted amounts of HDV RNA were quantified by RT-qPCR. Results showed that secretion of HDV RNA was decreased by GW4064 by 65% at 10 μM (FIG. 9A). This decrease of viral RNA was slightly more pronounced to that observed with interferon α-2a (50%). As expected, HBV polymerase inhibitor lamivudine, used as control, did not significantly modify HDV RNA secretion.

[0158] Then, to determine the specific infectivity of secreted HDV particles, concentrated HDV viral particles collected from dHepaRG supernatants were used to infect naïve Huh7.5-.sup.NTCP cells using the same vge/cell for each condition. Six days post infection, total intracellular HDV RNA was quantified by RT-qPCR. Results showed that, following infection with 500 vge/cell, the amount of intracellular HDV RNA was decreased by more than 95% in cells infected by supernatants obtained from dHepaRG treated with FXR agonist GW4064 (FIG. 9B) compared to a 70% decrease in the interferon α-2a condition. The specific infectivity of HDV particles was not modified by treatment with lamivudine.

[0159] Finally, naïve Huh7.5-.sup.NTCP cells were infected with the same concentrated supernatants but using two different HDV inoculum, 100 and 500 vge/cell, for each condition. Quantification of intracellular HDV RNAs 6 days post infection showed a dose dependent increase of HDV RNA levels in cells infected with supernatants collected from dHepaRG treated with either vehicle, interferon α-2a or lamivudine (FIG. 9C). However, this was not the case using supernatants collected from dHepaRG cells treated with FXR agonist GW4064, as no significant difference was observed when naïve Huh7.5-.sup.NTCP cells were infected with either 100 or 500 vge/cell. Overall, these results indicate that FXR agonist GW4064 severely decrease the infectious properties of secreted HDV particles.

CONCLUSIONS

[0160] The inventors found that FXR agonists are inhibitors of HDV replication in dHepaRG and PHH, the two most relevant models for in vitro studies of HDV infection. This antiviral effect was demonstrated with three different FXR agonists, i.e. one bile acid analog (6-ECDCA) and 2 synthetic agonists (GW4064 and tropifexor).

[0161] The present results from experiments performed in HDV monoinfected cells clearly demonstrate that the inhibitory effect of FXR agonists on HDV replication is independent of the impact of this class of molecules on HBV, which has been previously identified. Whereas HDV depends on HBV surface proteins for entry into hepatocytes, the replication step of the viral cycle occurs independently on HBV. As the inventors observed that the amount of the genomic form HDV RNA as well as nascent RNAs both decreased following treatment, FXR agonists may target the replication step of HDV life cycle.

[0162] Moreover, the inventors showed that FXR agonists are also inhibitors of HDV secretion and specific infectivity of secreted viral particles in dHepaRG cells. This antiviral effect was demonstrated with synthetic agonist GW4064.

[0163] In conclusion, the inventors have identified new molecules (i.e. FXR agonists) that specifically regulate (inhibit) HDV infection. This should allow the selection of candidates who could be tested in an animal model or directly in humans with FXR agonists already in clinical trials.

Material & Methods

Cell Lines

HepaRG

[0164] The HepaRG cell line derived from a human cellular hepato carcinoma can differentiate and regain many phenotypic traits of hepatocytes after 4 weeks of culture under defined conditions.sup.1. HepaRG cells were cultured, differentiated, and infected by HBV and HDV as previously described.sup.2,3. Briefly, for differentiation, cells were maintained for 2 weeks in standard medium then for at least 2 weeks in standard medium supplemented with 1.8% DMSO The composition of standard medium was the following: William's E medium supplemented with 10% HyCLone FetalClone II serum (Thermo Fisher Scientific), penicillin/streptomycin, L-glutamine, Insulin-Transferrin-Selenium (Gibco) and 50 μM hydrocortisone hemisuccinate.

Primary Human Hepatocytes

[0165] Primary human hepatocytes (PHH) were freshly prepared from human liver resection obtained from the Centre Leon Berard (Lyon) with French ministerial authorizations (AC 2013-1871, DC 2013-1870, AFNOR NF 96 900 sept 2011) as previously described.sup.4.

Huh7.5.SUP.NTCP

[0166] Huh7.5 cells were kindly provided by C. M. Rice (Rockefeller University, USA). Derived Huh7.5.sup.NTCP cells were generated by lentiviral transduction as previously described (Ni et al., Gastroenterology, 2014; 146(4):1070-83. doi: 10.1053/j.gastro.2013.12.024. Epub 2013 Dec 19. PMID: 24361467).

Viruses

[0167] HDV stocks (genotype 1, Genbank ID M21012) were prepared from supernatants from co-transfected Huh7 cells as previously described.sup.3,5. Plasmids pSVLD3 and pT7HB2.7 used for the production of infectious HDV particles have been kindly provided by Camille Sureau (Laboratoire de virologie moleculaire, Inserm UMR S_1134, Institut National de Transfusion Sanguine, Paris, France).

[0168] HBV stocks (genotype D, Genbank ID U95551) were prepared using the HepAD38 cell line according to previously described protocols.sup.7.

[0169] Supernatants containing HBV or HDV particles were clarified (0.45 μm filter) and concentrated with 8% PEG 8000 (Sigma-Aldrich).

[0170] HDV RNA was quantified by RT-qPCR as previously described.sup.6 and HBV DNA was quantified using the AmpliPrep/COBAS® TaqMan® HBV Test (Roche).

Chemicals

[0171] GW4064 [3-(2,6-dichlorophenyl)-4-(3-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl isoxazole] is a FXR agonist (EC50 90 nM), active both in vivo and in vitro.sup.8. Although displaying a limited bioavailability, GW4064 has gained a widespread use as a powerful and selective FXR agonist and has reached the status of “reference compound” in this field.

[0172] 6-ECDCA (6-ethyl-cheno-deoxycholic acide) is a bile salt derivative and strong FXR agonist (EC50 99 nM) and was obtained from Sigma-Aldrich.sup.9.

[0173] Tropifexor (2-[(1R,3r,5S)-3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]-1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3.2.1]octan-8-yl]-4-fluoro-1,3-benzothiazole-6-carboxylic acid) is a synthetic FXR agonist, active in vitro and in vivo, and was obtained from Cayman.sup.10.

[0174] GW4064, 6-ECDCA and tropifexor were all dissolved in DMSO at 10 mM to prepare stock solutions. Interferon alpha-2 (ROFERON-A) was purchased from Roche.

[0175] Actinomycin D was purchased from Sigma-Aldrich.

[0176] Lamivudine (LAM) was purchased from Selleckchem.

Western Blot

[0177] Cells were harvested in RIPA lysis buffer (NaCl 150 mM, Tris HCl pH=8.0 50 mM, SDS 0.1%, NP40 1%, Na Deoxycholate 0.5%) containing protease inhibitors (Protein Cocktail Inhibitors from Sigma-Aldrich, NaF 10 mM, Na Orthovanadate 10 mM). Clarified lysates were subjected to 10% SDS-PAGE and Western Blot transfer onto PVDF or nitrocellulose membranes using the TransTurbo Blot apparatus according to the manufacturer (Biorad). Primary antibodies are the HDVAg antibody (kind gift of Dr Alain Kay) and the beta-tubulin antibody (Abcam). Secondary HRP antibodies were purchased from Sigma-Aldrich. HRP signal detection was determined electronically using Ozyme—Syngene PXi Image system and parameters set strictly below the saturation point.

Northern Blot

[0178] Total RNA was extracted from infected cells using Tri Reagent® (TR118, Molecular Research Center). For each sample, 2 μg of total RNA were subjected to electrophoresis in gels of 1.2% agarose. After electrotransfer to charged nylon membranes (Roche), genomic HDV RNA sequences were detected by using a strand-specific RNA probe synthesized using the Dig RNA Labeling kit (Sp6/T7) (Roche) and DIG luminescent detection kit (Roche) accordinng to the manufacturer's instruction. Quantification of signals were done with ImageLab.

[0179] As an internal control for the amount and quality of the extracted RNA, the membrane was stripped and rehybridized by using labeled oligonucleotides specific for human 18S rRNA and 28S rRNA.

HBs and HBe Quantification

[0180] HBs and HBe antigens secreted in cells supernatant were quantified, after required dilutions, on Mini Vidas apparatus with Vidas HBs and Vidas HBE/HBET kits (bioMerieux, France) or Autobio kits (AutoBio, China) according to manufacturer's protocol.

Quantification of Viral RNAs by qPCR

[0181] Total RNA was prepared using NucleoSpin RNA Plus (Macherey-Nagel). After DNA digestion with TURBO DNase (Ambion), maximum 1000 ng RNA were reverse-transcribed using High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific). Quantitative PCR was carried out with primers HDV-F (5′-GCCTCTCCTTGTCGGTGAAT-3′, SEQ ID NO: 1) and HDV-R (5′-CCTGGCTGGGGAACATCAAA-3′, SEQ ID NO: 2) for quantification of total HDV RNA and HBV-F (5′-AGCTACTGTGGAGTTACTCTCGT-3′, SEQ ID NO: 3) and HBV-R (5′-CAAAGAATTGCTTGCCTGAGTG-3′, SEQ ID NO: 4) for quantification of pregenomic/precore HBV RNA. cDNA was analysed by quantitative PCR (qPCR) using QuantiFast SYBR® Green PCR kit (Qiagen) on LightCycler® 480 instrument (Roche) using a 45 PCR cycles. All assays were performed in triplicate. Relative quantification was determined by normalizing the expression of each gene to S9 housekeeping gene using primers S9-F (5′-CCGCGTGAAGAGGAAGAATG-3′, SEQ ID NO: 5) and S9-R (5′-TTGGCAGGAAAACGAGACAAT-3′, SEQ ID NO: 6).

Run-On Assays

[0182] HDV-infected HepaRG cells were incubated with labelled uridin or not (mock-EU) for 2 h, washed and harvested. Total intracellular HDV RNAs as well as EU-labelled HDV RNAs (nascent intracellular HDV RNAs) were isolated using the Click-iT™ Nascent RNA Capture Kit (Thermofisher Scientific) according to the manufacturer's instruction. As a control, cells were treated with 10 μg/mL of actinomycin D 20 min before incubation with labelled uridin in order to block transcription of nascent RNAs.

Analysis of HDV Secretion and Specific Infectivity

[0183] For analysis of HDV virion specific infectivity, supernatants from dHepaRG infected with both HBV and HDV were concentrated using 8% PEG 8000. HDV RNA was quantified by RT-qPCR in concentrates and Huh7.5.sup.NTCP cells were infected using same viral genome equivalents (vge) of concentrated virus for each condition of treatment. Six days post infection, total cellular RNA was extracted and HDV RNA was quantified by RT-qPCR.

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