T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR‐181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV‐infected individuals compared with age‐ and sex‐matched healthy subjects. Mechanistic studies revealed that up‐regulation of transcription factor ΔNp63 led to the decline of miR‐181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV‐infected individuals. Either reconstituting miR‐181a or silencing ΔNp63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence‐associated β‐galactosidase (SA‐β‐gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV‐induced T cell senescence is counterregulated by the ΔNp63–miR‐181a–Sirt1 pathway. An increase of IL‐2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3− effector T (Teff) cells upon manipulating the ΔNp63–miR‐181a–Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

Hepatitis C viral infection is the major cause of acute hepatitis and chronic liver disease and remains the leading cause of liver transplants (NIH). An estimated 180 million people are infected globally (WHO). There is no vaccine available to prevent hepatitis C. The treatment with antiviral drugs is expensive, accompanied with various side effects and is limited only to those at risk of developing advanced liver disease. The treatment is also effective in only about 30% to 50% of treated patients and still a high percentage of patients are resistant to therapy. Therefore, there is an urgent need for the development of effective vaccine antigens and an efficacious HCV vaccine. The non-structural 3 protein of the hepatitis C virus is a multifunctional protein of the virus required for virus polyprotein processing and replication. Vaccine antigen production via chloroplast transformation system usually results in high expression levels and eliminates the possibility of contamination with vector sequences,human or animal pathogens. The HCV NS3 antigen was expressed in the chloroplast of Nicotiana tabacum var. Petit havana and LAMD-609. The 1.9kb NS3 gene was cloned into a chloroplast expression vector, pLD-Ct containing the 16S rRNA promoter, aadA gene coding for the spectinomycin selectable marker, psbA 5' untranslated region to enhance translation in the light and 3' untranslated region for transcript stability and trnI & trnA homologous flanking sequences for site specific integration into the chloroplast genome. Chloroplast integration of the NS3 gene was first confirmed by PCR. Southern blot analysis further confirmed site-specific gene integration and homoplasmy. The NS3 protein was detected in transgenic chloroplasts by immunoblot analysis. The NS3 protein was further quantified by ELISA. Maximum expression levels of NS3 up to 2% in the total soluble protein were observed even in old leaves, upon 3-day continuous illumination. These results demonstrate successful expression of the HCV non-structural 3 antigen in transgenic tobacco chloroplasts. Animal studies to test the immunogenecity of the chloroplast derived HCV NS3 will be performed using chloroplast derived NS3 antigen.

In 2014, an analysis was conducted to evaluate the hepatitis C virus (HCV) epidemiology and disease burden in Germany. Since then, there have been considerable developments in HCV management such as the implementation of direct acting antivirals. The aim of this analysis was to assess the recent data available for Germany, establish an updated 2020 HCV prevalence and cascade of care and evaluate the impact of what-if scenarios on the future burden of disease using modelling analysis. A dynamic Markov model was used to forecast the HCV disease burden in Germany. Model inputs were retrieved through literature review, unpublished sources and expert input. Next, three “what-if” scenarios were developed to evaluate the status quo, COVID-19 pandemic, and steps needed to achieve the WHO targets for elimination. At the beginning of 2020, there were 189,000 (95% UI: 76,700–295,000) viremic infections in Germany, a decline of more than 85,000 viremic infections since 2012. Annual treatment starts went down since 2015. Compared with 2019, the COVID-19 pandemic resulted in a further 11% decline in 2020. If this continues for two years, it could result in 110 excess HCC cases and 200 excess liver related deaths by 2030. To achieve the WHO targets, 81,200 people need to be diagnosed, with 118,600 initiated on treatment by 2030. This could also avert 1,020 deaths and 720 HCC cases between 2021 and 2030. Germany has made strides towards HCV elimination, but more efforts are needed to achieve the WHO targets by 2030.

No description available.

As obligatory intracellular parasites, viruses are in a constant battle with their host to establish infection. They can facilitate their propagation by modulating host immune or metabolic pathways. This modulation involves targeting various molecular factors such as microRNAs (miRNA), enzymes, or small molecules. Understanding how viruses alter the chemical makeup of a cell is crucial to identifying what pathways are being targeted, furthering our understanding of the virus life cycle, and may aid in identifying biomarkers of disease. Here, we examine host-virus interactions in the context of two viruses, hepatitis c virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). First, the modulation of serine hydrolases by a pro-viral microRNA, miRNA-122, is investigated using activity-based protein profiling (ABPP). This study identifies a downstream target of miRNA-122 that is differentially activated during HCV infection which can be targeted pharmacologically to reduce HCV infectivity. Second, we apply similar techniques to identify serine hydrolase changes associated with SARS-CoV-2 infection. Results point towards enrichment of endocannabinoid metabolism which may offer an alternative therapeutic avenue for combating SARS-CoV-2 infection. Together, the work presented in this thesis provides avenues for further investigation into miRNA-122 interactions during HCV infection and endocannabinoid metabolism in SARS-CoV-2 infection.

No description available.

Hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma worldwide. HCV-neutralizing antibody AP33 recognizes a linear, highly conserved epitope on the viral entry protein E2, disrupting the interaction with the cellular receptor CD81 that leads to viral entry. AP33-related anti-idiotypes (Ab₂s) have the potential to carry the internal image of the antigen E2, eliciting the production of AP33-like antibodies in humans. This study reports the mid-resolution structure of the Fab fragment of anti-idiotype A164.3 and the high-resolution structure of the Fab fragment of AP33 in complex with the Fv fragment of anti-idiotype B2.1A. Analysis of the structures and comparison with the previously published structure of AP33 in complex with a peptide corresponding to the E2 epitope, suggests that while A164.3 does not mimic the antigen E2, B2.1A is characterized by high surface complementarity with AP33 and functional antigen mimicry. Thus, B2.1A can be classified as an Ab₂-β, a subgroup of anti-idiotypes carrying the internal image of the antigen. Preliminary binding studies show that AP33 binds B2.1A with nanomolar affinity, supporting the role of B2.1A as an idiotypic vaccine candidate. Zaire ebola virus causes severe, often lethal hemorrhagic fever in humans. Ebola virus polymerase cofactor VP35 is a multifunctional protein involved in, among other functions, dsRNA binding and inhibition of the host's interferon pathways. VP35 contains an N-terminal oligomerization domain and a C-terminal dsRNA-binding domain (RBD). Preliminary results on the oligomerization domain of VP35 suggest that this region contains a coiled-coil motif, as previously reported. In order to validate a recently-discovered dsRNA end-capping pocket as a drug target, the structure of VP35 RBD I278A mutant was solved at high resolution, showing that even a small perturbation in the binding pocket can cause dramatic binding impairment due to loss of contacts with dsRNA.

[Truncated abstract] Around 50,000 hip and knee replacements are performed every year in Australia and this number has been increasing by around 13% annually since 1998 (Transplantation Society 2006). The incidence and number of revision surgery has increased by a similar proportion. Autogenous bone or allograft is still the gold standard grafting material and is currently used in a variety of reconstructive surgical procedures. The use of any allograft material carries with it the risk of transfer of disease from donor to recipient. These tissues can transmit the same viral and bacterial infections as blood, and the products of a single donation may be transplanted to several recipients. In contrast to blood, musculoskeletal tissues may come from surgical and cadaveric donation. Overall, the prevention of infection relies on the maintenance of rigid protocols for procurement, donor and allograft testing, secondary sterilisation, and the adherence to internal safety standards within the tissue banks. This thesis aims to determine the risk of viral infection among musculoskeletal tissue donors in Australia. We retrieved and analysed data retrospectively from three large tissue banks in Australia (Perth, Queensland, Victoria). This includes 12,415 musculoskeletal tissue donors, 10,937 of which are surgical donors and 1,478 of which are deceased donors, for the period of 1993 -2004. This data was analysed to determine the prevalence and incidence of viral infections such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T-lymphotropic virus (HTLV) in musculoskeletal allografts. The results indicate that the risk of viral infection from musculoskeletal tissue transplantation in Australia is low. ... The results indicate that the overall prevalence of screened transfusion-transmitted viral infections did not vary significantly for musculoskeletal donors over the study period, despite falling in the general population and first-time blood donors. In tissue donors, HIV incidence significantly decreased over time, and HBV decreased significantly during 1999-2001; however, there was an apparent increase in the estimated incidence of HCV in 2002-2004 compared with earlier years. Furthermore the residual risk estimate of HIV in the period 2002-2004 has declined 5-fold compared to estimates in the period 1993-1995. This is perhaps due to greater awareness of high risk behaviours among donors, improvement in donor recruitment and an overall decrease in infection levels in the general population. Musculoskeletal tissue is second only to blood as the most frequent transplanted human tissue. Viral infection is a potential complication of tissue transplantation. In this thesis the rates of HIV, HBV, HCV and HTV infection in musculoskeletal donors in Australia were identified and then compared with results in published data from Canada, Scotland and the United States. The study also compared that result with first-time blood donors because they have satisfied similar donor selection criteria (Galea et al. 2006). The results indicate that prevalence and incidence estimates for viral infection in Australian tissue donors are higher than those in blood donors. This was also reported in studies from other countries. Accordingly, it is crucial that viral prevalence and incidence be monitored to evaluate the safety of tissue supply and to improve donor selection processes.

Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions.

The current treatment for hepatitis C virus (HCV) infection – combination therapy with pegylated interferon and ribavirin – elicits sustained responses in only ~50% of the patients treated. Greater cumulative exposure to ribavirin increases response to interferon-ribavirin combination therapy. A key limitation, however, is the toxic sideeffect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan in patients and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones. A small fraction of the population (~30%) with polymorphisms in the ITPA gene shows protection from ribavirin-induced anemia. The optimum dosage of ribavirin that can be tolerated is then dependent on the ITPA polymorphisms. Coupled with a previous population pharmacokinetic study, our model yields a facile formula for estimating the optimum dosage given a patient’s weight, creatinine clearance, pretreatment hemoglobin levels, and ITPA polymorphism. The reduced lifespan we predict is in agreement with independent measurements from breath tests as well as estimates derived from in vitro studies of ATP depletion. The latter estimates also agree with the extent of ATP depletion due to ribavirin that we predict from a detailed analysis of the nucleoside metabolism in erythrocytes. Our model thus facilitates in conjunction with models of viral kinetics the rational identification of treatment protocols. Our formula for optimum dose presents an avenue for personalizing ribavirin dosage. By keeping anemia tolerable, the predicted optimal dosage may improve adherence, reduce the need for drug monitoring, and increase response rates.