Point of care testing for hepatitis C in the priority settings of mental health, prisons and drug & alcohol facilities - the PROMPt Study.

BACKGROUND: A barrier to hepatitis C virus (HCV) cure is conventional testing. The aim of this study was to evaluate the effect of HCV antibody and RNA point-of-care-testing (POCT) on testing rates, linkage to care, treatment and acceptability of testing in three priority settings in Australia. METHODS: Participants were enrolled in an interventional cohort study at a reception prison, inpatient mental health service (MHS), and inpatient alcohol and other drug (AOD) unit-between October 2020 and December 2021. HCV POCT was performed using SD Bioline HCV antibody fingerstick test and a reflexive Xpert® HCV Viral Load Fingerstick test using capillary blood samples. A retrospective audit of HCV testing and treatment data was performed at each site for the preceding 12-month period to generate a historical control. RESULTS: 1,549 participants received a HCV antibody test with 17% (264/1,549) receiving a positive result, of which 21% (55/264) tested HCV RNA positive. Across all settings the rate of testing per year significantly increased between the historical controls and the study intervention period by three-fold (RR:2.57 95% CI: 2.32, 2.85) for HCV antibody testing and four-fold (RR:1.62; 95% CI:1.31, 2.01) for RNA testing. Treatment uptake was higher during the POCT intervention (86%, 47/55; P=0.010) compared to the historical controls (61%, 27/44). CONCLUSIONS: This study demonstrated across three settings that the use of HCV antibody and RNA POCT increased testing rates, treatment uptake linkage to care. The testing model was highly acceptable for most participants. CLINICAL TRIAL REGISTRATION: ACTRN-12621001578897.

Assessment of hepatitis B virus infection and interhost cellular responses using intrahepatic cholangiocyte organoids.

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma-derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor-dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor-dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor-dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.

SARS-CoV-2 Omicron variant escapes neutralizing antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry.

The interferon-induced transmembrane (IFITM) family of proteins have recently been identified as important host effector molecules of the type I interferon response against viruses. IFITM1 has been identified as a potent antiviral effector against hepatitis C virus (HCV), whereas the related family members IFITM2 and IFITM3 have been described to have antiviral effects against a broad range of RNA viruses. Here, we demonstrate that IFITM2 and IFITM3 play an integral role in the interferon response against HCV and act at the level of late entry stages of HCV infection. We have established that in hepatocytes, IFITM2 and IFITM3 localize to the late and early endosomes, respectively, as well as the lysosome. Furthermore, we have demonstrated that S-palmitoylation of all three IFITM proteins is essential for anti-HCV activity, whereas the conserved tyrosine residue in the N-terminal domain of IFITM2 and IFITM3 plays a significant role in protein localization. However, this tyrosine was found to be dispensable for anti-HCV activity, with mutation of the tyrosine resulting in an IFITM1-like phenotype with the retention of anti-HCV activity and co-localization of IFITM2 and IFITM3 with CD81. In conclusion, we propose that the IFITM proteins act in a coordinated manner to restrict HCV infection by targeting the endocytosed HCV virion for lysosomal degradation and demonstrate that the actions of the IFITM proteins are indeed virus and cell-type specific.

Fatty Acids Induce a Pro-Inflammatory Gene Expression Profile in Huh-7 Cells That Attenuates the Anti-HCV Action of Interferon.

The pathogenesis of nonalcoholic steatohepatitis is primarily an immune-driven disease and a known factor associated with treatment failure of chronic hepatitis C with interferon (IFN) and ribavirin. We studied the hepatocyte response in a model of steatosis at the transcriptome level and the antiviral action of IFN against hepatitis C virus (HCV) in this setting. In this study, we have shown that lipid loading (oleic acid and palmitic acid, OA:PA) of Huh-7 cells leads to increased expression of classical interferon-stimulated genes (ISGs) and NF-κß-dependent pro-inflammatory genes. A selective blocker of Toll-like receptor (TLR)2 signaling suppressed NF-κß promoter activity by OA:PA, suggesting that free fatty acids (FFAs) act as a TLR2 pathogen-associated molecular pattern. Furthermore, in the presence of OA:PA, IFN stimulation and HCV infection (Jc1) increased ISG expression. Somewhat counterintuitive to the increase in ISGs, the anti-HCV activity of IFN was attenuated in the presence of OA:PA. Interestingly, the combination of OA:PA, HCV, and IFN-α stimulation resulted in a significant increase in CXCL8 protein production, a cytokine known to have anti-IFN modulating activity. Thus, in an in vitro model of steatosis, the FFAs OA and PA drive an NF-κß-dependent inflammatory and ISG gene expression profile via TLR2 activation. Furthermore, FFA synergistically increases IFN-driven gene expression that may account for HCV treatment failure in vivo.

Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection.

UNLABELLED: Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways. IMPORTANCE: Hepatitis C virus (HCV) is a major cause of serious liver disease worldwide. An improved understanding of the HCV replication cycle will enable development of novel and improved antiviral strategies. Here we have developed complementary fluorescent labeling and imaging approaches to investigate the localization, traffic and interactions of the HCV NS5A protein in living, virus-producing cells. These studies reveal new details as to the traffic, composition and biogenesis of NS5A foci and the nature of their association with putative sites of virus assembly.

Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus.

UNLABELLED: Host factors play an important role in all facets of the hepatitis C virus (HCV) life cycle and one such host factor is signal transducer and activator of transcription 3 (STAT3). The HCV core protein has been shown to directly interact with and activate STAT3, while oxidative stress generated during HCV replication in a replicon-based model also induced STAT3 activation. However, despite these findings the precise role of STAT3 in the HCV life cycle remains unknown. We have established that STAT3 is actively phosphorylated in the presence of replicating HCV. Furthermore, expression of a constitutively active form of STAT3 leads to marked increases in HCV replication, whereas, conversely, chemical inhibition and small interfering RNA (siRNA) knockdown of STAT3 leads to significant decreases in HCV RNA levels. This strongly implicates STAT3 as a proviral host factor. As STAT3 is a transcription factor, up-regulation of a distinct set of STAT3-dependent genes may create an environment that is favorable for HCV replication. However, STAT3 has recently been demonstrated to positively regulate microtubule (MT) dynamics, by way of a direct sequestration of the MT depolymerizing protein Stathmin 1 (STMN1), and we provide evidence that STAT3 may exert its effect on the HCV life cycle by way of positive regulation of MT dynamics. CONCLUSION: We have demonstrated that STAT3 plays a role in the life cycle of HCV and have clarified the role of STAT3 as a proviral host factor.

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.

UNLABELLED: The interferon-stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell-culture-derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid-droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle-associated membrane protein-associated protein subtype A (VAP-A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus, as well as an N-terminal amphipathic helix. Removal of the amphipathic helix-redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis. CONCLUSION: In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP-A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon-stimulated gene expression.

Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific.

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.

Impact of alcohol on hepatitis C virus replication and interferon signaling.

Hepatitis C virus (HCV) is one of the main etiological factors responsible for liver disease worldwide. It has been estimated that there are over 170 million people infected with HCV worldwide. Of these infected individuals, approximately 75% will go on to develop a life long necroinflammatory liver disease, which over decades, can result in serious complications, such as cirrhosis and hepatocellular carcinoma. Currently there is no effective vaccine and whilst antiviral therapies have been improved, they are still only effective in approximately 50% of individuals. HCV infection stands as a major cause of global morbidity and suffering, and places a significant burden on health systems. The second highest cause of liver disease in the western world is alcoholic liver disease. Frequently, HCV infected individuals consume alcohol, and the combined effect of HCV and alcohol consumption is deleterious for both liver disease and response to treatment. This review discusses the impact of alcohol metabolism on HCV replication and the negative impact on interferon (IFN)-alpha treatment, with a particular focus on how alcohol and HCV act synergistically to increase oxidative stress, ultimately leading to exacerbated liver disease and a reduction in the efficacy of IFN-alpha treatment. A better understanding of the complicated mechanisms at play in hepatocytes infected with HCV and metabolizing alcohol will hopefully provide better treatment options for chronic hepatitis C individuals that consume alcohol.

Alcohol metabolism increases the replication of hepatitis C virus and attenuates the antiviral action of interferon.

The interactions between hepatitis C virus (HCV) and alcohol metabolism are not well understood. To determine the effect that alcohol metabolism has on HCV replication and the antiviral action of interferon (IFN), Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of cytochrome P450 2E1 (Cyp2e1) were treated with ethanol and IFN-alpha. Treatment of these cells with ethanol (0-100 mmol/L) significantly increased HCV replication. This effect was dependent on Cyp2e1 expression and alcohol-metabolized oxidative stress (OS), because the antioxidant N-acetylcysteine blocked this effect. Furthermore, the anti-HCV action of IFN-alpha was attenuated in the presence of ethanol metabolism, most likely via attenuation of Stat1 tyrosine-701 phosphorylation. These in vitro results mimic what is often noted clinically, and further dissection of this model system will aid in our understanding of interactions between HCV and alcohol metabolism.

A screening method for identifying disruptions in interferon signaling reveals HCV NS3/4a disrupts Stat-1 phosphorylation.

UNLABELLED: Viruses have evolved mechanisms to inhibit the innate immune response to infection. The aim of this study was to develop an efficient screening method to identify viral proteins and their ability to block Jak-Stat signaling using hepatitis C virus (HCV) as an example. The 2FTGH cell assay system was used in combination with transient transfection of HCV proteins in this study. Using 1000U/ml IFN and 30mM 6-TG to treat 2FTGH cells, it was established that transient protein expression in this cell system yielded 39% and 0% cell survival for the positive (HPV E7) and negative controls (GFP expression) respectively. Transient expression of HCV Core-p7 resulted in 22% cell survival, consistent with previous reports, while expression of the HCV serine protease NS3/4a resulted in 54% cell survival. NS3/4a was subsequently shown to inhibit phosphorylation of Stat-1 at the serine residue 727. CONCLUSION: the 2FTGH cell assay system can be adapted for transient screening to examine the ability of viral proteins or other potential inhibitors to block the Jak-Stat signaling pathway. We show that HCV NS3/4a is able to block this pathway at the stage of Stat-1 serine 727 phosphorylation.