Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Abstract Background About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Computational investigations of ion conduction mechanisms using enhanced sampling methods

Proper ion channels’ functioning is a prerequisite for a normal cell and disorders involving ion channels, or channelopathies, underlie many human diseases. Long QT syndromes (LQTS) for example may arise from the malfunctioning of hERG channel, caused either by the binding of drugs or mutations in HERG gene. In the first part of this thesis I present a framework to investigate the mechanism of ion conduction through hERG channel. The free energy profile governing the elementary steps of ion translocation in the pore was computed by means of umbrella sampling simulations. Compared to previous studies, we detected a different dynamic behavior: according to our data hERG is more likely to mediate a conduction mechanism which has been referred to as “single-vacancy-like” by Roux and coworkers (2001), rather then a “knock-on” mechanism. The same protocol was applied to a model of hERG presenting the Gly628Ser mutation, found to be cause of congenital LQTS. The results provided interesting insights about the reason of the malfunctioning of the mutant channel. Since they have critical functions in viruses’ life cycle, viral ion channels, such as M2 proton channel, are considered attractive targets for antiviral therapy. A deep knowledge of the mechanisms that the virus employs to survive in the host cell is of primary importance in the identification of new antiviral strategies. In the second part of this thesis I shed light on the role that M2 plays in the control of electrical potential inside the virus, being the charge equilibration a condition required to allow proton influx. The ion conduction through M2 was simulated using metadynamics technique. Based on our results we suggest that a potential anion-mediated cation-proton exchange, as well as a direct anion-proton exchange could both contribute to explain the activity of the M2 channel.

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Lo sviluppo di disturbi psichiatrici in pazienti hcv+ sottoposti a terapia antivirale: L'impatto dei nuovi farmaci

Introduzione:l’interferone (IFN) usato per l’eradicazione del virus dell’Epatite C, induce effetti collaterali anche riferibili alla sfera psichica. I dati sugli eventi avversi di tipo psichiatrico dei nuovi farmaci antivirali (DAA) sono limitati. Lo scopo di questo studio è di valutare lo sviluppo di effetti collaterali di tipo psichiatrico in corso di due distinti schemi di trattamento: IFN-peghilato e ribavirina [terapia duplice (standard o SOC)]; DAA in associazione a IFN-peghilato e ribavirina (terapia triplice). Metodi: pazienti HCV+ consecutivi seguiti presso l’Ambulatorio delle Epatiti Croniche della Semeiotica Medica del Dipartimento di Scienze Mediche e Chirurgiche dell’Università di Bologna in procinto di intraprendere un trattamento antivirale a base di IFN, sottoposti ad esame psicodiagnostico composto da intervista clinica semistrutturata e test autosomministrati: BDI, STAXI-2, Hamilton Anxiety Scale, MMPI – 2. Risultati: Sono stati arruolati 84 pazienti, 57/84 (67.9%) nel gruppo in triplice e 27/84 nel gruppo SOC. Quasi tutti i pazienti arruolati hanno eseguito l’intervista clinica iniziale (82/84; 97.6%), mentre scarsa è stata l’aderenza ai test (valori missing>50%). Ad eccezione dell’ansia, la prevalenza di tutti gli altri disturbi (irritabilità, astenia, disfunzioni neurocognitive, dissonnia) aumentava in corso di trattamento. In corso di terapia antivirale 43/84 (51.2%) hanno avuto bisogno di usufruire del servizio di consulenza psichiatrica e 48/84 (57.1%) hanno ricevuto una psicofarmacoterapia di supporto, senza differenze significative fra i due gruppi di trattamento. Conclusioni : uno degli elementi più salienti dello studio è stata la scarsa aderenza ai test psicodiagnostici, nonostante l’elevata prevalenza di sintomi psichiatrici. I risultati di questo studio oltre ad evidenziare l’importanza dei sintomi psichiatrici in corso di trattamento e la rilevanza della consulenza psicologica e psichiatrica per consentire di portare a termine il ciclo terapeutico previsto (migliorandone l’efficacia), ha anche dimostrato che occorre ripensare gli strumenti diagnostici adattandoli probabilmente a questo specifico target.

Influenza A(H1N1) infection and severe cardiac dysfunction in adults: A case series

BACKGROUND: While viral myocarditis and heart failure are recognized and feared complications of seasonal influenza A infection, only limited information is available for 2009 influenza A(H1N1)-induced heart failure. METHODS AND MAIN FINDINGS: This case series summarizes the disease course of four patients with 2009 influenza A(H1N1) infection who were treated at our institution from November 2009 until September 2010. All patients presented with severe cardiac dysfunction (acute heart failure, cardiogenic shock or cardiac arrest due to ventricular fibrillation) as the leading symptom of influenza A(H1N1) infection. Two patients most likely had pre-existent cardiac pathologies, and three required catecholamine therapy to maintain hemodynamic function. Except for one patient who died before influenza A(H1N1) infection had been diagnosed, all patients received antiviral therapy with oseltamivir and supportive critical care. Acute respiratory distress syndrome due to influenza A(H1N1) infection developed in one patient. Heart function normalized in two of the three surviving patients but remained impaired in the other one at hospital discharge. CONCLUSIONS: Influenza A(H1N1) infection may be associated with severe cardiac dysfunction which can even be the leading clinical symptom at presentation. During an influenza pandemic, a thorough history may reveal flu-like symptoms and should indicate testing for H1N1 infection also in critically ill patients with acute heart failure.

Prevalence of comedications and effect of potential drug-drug interactions in the Swiss HIV Cohort Study

Potential drug-drug interactions (PDDIs) might expand with new combination antiretroviral therapies (ART) and polypharmacy related to increasing age and comorbidities. We investigated the prevalence of comedications and PDDIs within a large HIV cohort, and their effect on ART efficacy and tolerability.

Eligibility for and outcome of hepatitis C treatment of HIV-coinfected individuals in clinical practice: the Swiss HIV cohort study

BACKGROUND: Morbidity and mortality of individuals co-infected with HIV and hepatitis C virus (HCV) is often determined by the course of their HCV infection. Only a selected proportion of those in need of HCV treatment are studied in randomized controlled trials (RCTs). We analysed the prevalence of HCV infection in a large cohort, the number of individuals requiring treatment, the eligibility for HCV treatment, and the outcome of the combination therapy with pegylated interferon-a and ribavirin in routine practice. METHODS: We analysed prescription patterns of HCV treatment and treatment outcomes among participants from the Swiss HIV Cohort Study with detectable hepatitis C viraemia (between January 2001 and October 2004). Efficacy was measured by the number of patients with undetectable HCV RNA at the end of therapy (EOTR) and at 6 months after treatment termination (SVR). Intention-to-continue-treatment principles were used. RESULTS: A total of 2150 of 7048 (30.5%) participants were coinfected with HCV; HCV RNA was detected in 60%, and not assessed in 26% of HCV-antibody-positive individuals. One hundred and sixty (12.5%) of HCV-RNA-positive patients started treatment. In patients infected with HCV genotypes 1/4 or 2/3, EOTR was achieved in 43.3% and 81.2% of patients, respectively, and SVR rates were 28.4% and 51.8%, respectively. More than 50% of the HCV-treated patients would have been excluded from two large published RCTs due to demographic, clinical and laboratory criteria. CONCLUSIONS: Despite clinical and psychosocial obstacles encountered in clinical practice, HCV treatment in HIV-coinfected individuals is feasible with results similar to those obtained in RCTs.

Adverse events to antiretrovirals in the Swiss HIV Cohort Study: effect on mortality and treatment modification

BACKGROUND: Antiretroviral therapy (ART) decreases morbidity and mortality in HIV-infected patients but is associated with considerable adverse events (AEs). METHODS: We examined the effect of AEs to ART on mortality, treatment modifications and drop-out in the Swiss HIV Cohort Study. A cross-sectional evaluation of prevalence of 13 clinical and 11 laboratory parameters was performed in 1999 in 1,078 patients on ART. AEs were defined as abnormalities probably or certainly related to ART. A score including the number and severity of AEs was defined. The subsequent progression to death, drop-out and treatment modification due to intolerance were evaluated according to the baseline AE score and characteristics of individual AEs. RESULTS: Of the 1,078 patients, laboratory AEs were reported in 23% and clinical AEs in 45%. During a median follow up of 5.9 years, laboratory AEs were associated with higher mortality with an adjusted hazard ratio (HR) of 1.3 (95% confidence interval [CI] 1.2-1.5; P < 0.001) per score point. For clinical AEs no significant association with increased mortality was found. In contrast, an increasing score for clinical AEs (HR 1.11,95% CI 1.04-1.18; P = 0.002), but not for laboratory AEs (HR 1.07, 95% CI 0.97-1.17; P = 0.17), was associated with antiretroviral treatment modification. AEs were not associated with a higher drop-out rate. CONCLUSIONS: The burden of laboratory AEs to antiretroviral drugs is associated with a higher mortality. Physicians seem to change treatments to relieve clinical symptoms, while accepting laboratory AEs. Minimizing laboratory drug toxicity seems warranted and its influence on survival should be further evaluated.

Tenofovir use is associated with a reduction in calculated glomerular filtration rates in the Swiss HIV Cohort Study

BACKGROUND: A growing number of case reports have described tenofovir (TDF)-related proximal renal tubulopathy and impaired calculated glomerular filtration rates (cGFR). We assessed TDF-associated changes in cGFR in a large observational HIV cohort. METHODS: We compared treatment-naive patients or patients with treatment interruptions > or = 12 months starting either a TDF-based combination antiretroviral therapy (cART) (n = 363) or a TDF-sparing regime (n = 715). The predefined primary endpoint was the time to a 10 ml/min reduction in cGFR, based on the Cockcroft-Gault equation, confirmed by a follow-up measurement at least 1 month later. In sensitivity analyses, secondary endpoints including calculations based on the modified diet in renal disease (MDRD) formula were considered. Endpoints were modelled using pre-specified covariates in a multiple Cox proportional hazards model. RESULTS: Two-year event-free probabilities were 0.65 (95% confidence interval [CI] 0.58-0.72) and 0.80 (95% CI 0.76-0.83) for patients starting TDF-containing or TDF-sparing cART, respectively. In the multiple Cox model, diabetes mellitus (hazard ratio [HR] = 2.34 [95% CI 1.24-4.42]), higher baseline cGFR (HR = 1.03 [95% CI 1.02-1.04] by 10 ml/min), TDF use (HR = 1.84 [95% CI 1.35-2.51]) and boosted protease inhibitor use (HR = 1.71 [95% CI 1.30-2.24]) significantly increased the risk for reaching the primary endpoint. Sensitivity analyses showed high consistency. CONCLUSION: There is consistent evidence for a significant reduction in cGFR associated with TDF use in HIV-infected patients. Our findings call for a strict monitoring of renal function in long-term TDF users with tests that distinguish between glomerular dysfunction and proximal renal tubulopathy, a known adverse effect of TDF.

Late presentation of HIV-infected individuals

Late presentation remains a major concern despite the dramatically improved prognosis realized by ART. We define a first presentation for HIV care during the course of HIV infection as 'late' if an AIDS-defining opportunistic disease is apparent, or if CD4+ T-cells are <200/microl. In the Western world, approximately 10 and 30% of HIV-infected individuals still present with CD4+ T-cells <50 and <200/microl, respectively; estimates are substantially higher for developing countries. Diagnosis and treatment of opportunistic diseases and intense supportive in-hospital care take precedence over ART. Benefits of starting ART without delay, that is, when opportunistic diseases are still active, include faster resolution of opportunistic diseases and a decreased risk of recurrence. The downside of starting ART without delay could include toxicity, drug interactions and immune reconstitution inflammatory syndrome (IRIS). Among asymptomatic or oligosymptomatic individuals presenting late, where ART and primary prophylaxis are initiated, approximately 10-20% will become symptomatic from drug toxicity or undiagnosed opportunistic complications, including IRIS, which require appropriate therapies. In this review we describe late presentation to HIV care, the scale of the problem, the evaluation of a late-presenting patient and challenges associated with initiation of potent antiretroviral therapy (ART) in the setting of acute opportunistic infections and other comorbidities.

Does tenofovir influence efavirenz pharmacokinetics?

INTRODUCTION: A recent report described a possible interaction between tenofovir (TFV) and efavirenz (EFV). Patients developed neuropsychiatric manifestations upon introduction of TFV on a stable EFV-containing regimen. We evaluated the possibility of a pharmacokinetic interaction between TFV and EFV by assessing cross-sectional and longitudinal data in 169 individuals receiving EFV. RESULTS: EFV plasma area-under-the-curve (AUC) levels were comparable among individuals receiving (n=18) or not receiving TFV (n=151); 57,962 versus 52,293 ng*h/ml. However, under conditions of limited EFV metabolism, that is, the group of 23 individuals carrying two copies of CYP2B6 loss/diminished-function alleles, plasma AUC values were highest among individuals receiving TFV (n=5, 353,031 ng*h/ml), compared with those not receiving TFV (n=18, 180,689 ng*h/ml). Statistical analysis identified both a global, sixfold effect of CYP2B6 loss/diminished function (P < 0.0001) and a significant interaction between the number of loss/diminished-function alleles and the co-medication with TFV (P = 0.009). CONCLUSION: Although there is no clear evidence for a pharmacokinetic interaction between TFV and EFV, we cannot rule out an interaction between these drugs restricted to individuals who are slow EFV metabolizers.

Body fat changes among antiretroviral-naive patients on PI- and NNRTI-based HAART in the Swiss HIV cohort study

BACKGROUND: Body fat changes are common in patients with HIV. For patients on protease inhibitor (PI)-based highly active antiretroviral therapy (HAART), these changes have been associated with increasing exposure to therapy in general and to stavudine in particular. Our objective is to show whether such associations are more or less likely for patients on non-nucleoside reverse transcriptase inhibitor (NNRTI)-based HAART. METHODS: We included all antiretroviral-naive patients in the Swiss HIV Cohort Study starting HAART after April 2000 who had had body weight, CD4 cell count and plasma HIV RNA measured between 6 months before and 3 months after starting HAART, and at least one assessment of body fat changes after starting HAART. At visits scheduled every 6 months, fat loss or fat gain is reported by agreement between patient and physician. We estimate the association between reported body fat changes and both time on therapy and time on stavudine, using conditional logistical regression. RESULTS: Body fat changes were reported for 85 (9%) out of 925 patients at their first assessment; a further 165 had only one assessment. Of the remaining 675 patients, body fat changes were reported for 156 patients at a rate of 13.2 changes per 100 patient-years. Body fat changes are more likely with increasing age [odds ratio (OR) 1.18 (1.00-1.38) per 10 years], with increasing BMI [OR 1.06 (1.01-1.11)] and in those with a lower baseline CD4 cell count [OR 0.91 (0.83-1.01) per 100 cells/microl]. There is only weak evidence that body fat changes are more likely with increasing time on HAART [OR 1.16 (0.93-1.46)]. After adjusting for time on HAART, fat loss is more likely with increasing stavudine use [OR 1.70 (1.34-2.15)]. There is no evidence of an association between reported fat changes and time on NNRTI therapy relative to PI therapy in those patients who used either one therapy or the other [OR 0.98 (0.56-1.63)]. CONCLUSION: Fat loss is more likely to be reported with increasing exposure to stavudine. We find no evidence of major differences between PI and NNRTI therapy in the risk of reported body fat changes.