Leda Bassit

Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease

Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals.

Hepatitis B virus infection in Haemodialysis Centres from Santa Catarina State, Southern Brazil. Predictive risk factors for infection and molecular epidemiology

BackgroundPatients under haemodialysis are considered at high risk to acquire hepatitis B virus (HBV) infection. Since few data are reported from Brazil, our aim was to assess the frequency and risk factors for HBV infection in haemodialysis patients from 22 Dialysis Centres from Santa Catarina State, south of Brazil.MethodsThis study includes 813 patients, 149 haemodialysis workers and 772 healthy controls matched by sex and age. Serum samples were assayed for HBV markers and viraemia was detected by nested PCR. HBV was genotyped by partial S gene sequencing. Univariate and multivariate statistical analyses with stepwise logistic regression analysis were carried out to analyse the relationship between HBV infection and the characteristics of patients and their Dialysis Units.ResultsFrequency of HBV infection was 10.0%, 2.7% and 2.7% among patients, haemodialysis workers and controls, respectively. Amidst patients, the most frequent HBV genotypes were A (30.6%), D (57.1%) and F (12.2%). Univariate analysis showed association between HBV infection and total time in haemodialysis, type of dialysis equipment, hygiene and sterilization of equipment, number of times reusing the dialysis lines and filters, number of patients per care-worker and current HCV infection. The logistic regression model showed that total time in haemodialysis, number of times of reusing the dialysis lines and filters, and number of patients per worker were significantly related to HBV infection.ConclusionsFrequency of HBV infection among haemodialysis patients at Santa Catarina state is very high. The most frequent HBV genotypes were A, D and F. The risk for a patient to become HBV positive increase 1.47 times each month of haemodialysis; 1.96 times if the dialysis unit reuses the lines and filters ≥ 10 times compared with haemodialysis units which reuse < 10 times; 3.42 times if the number of patients per worker is more than five. Sequence similarity among the HBV S gene from isolates of different patients pointed out to nosocomial transmission.

HCV drug discovery aimed at viral eradication

Summary.  Hepatitis C virus (HCV) causes significant morbidity and mortality worldwide with nearly 3% of the world population infected by this virus. Fortunately, this virus does not establish latency, and hence it may be possible to eradicate it. HCV is strongly associated with liver cirrhosis and hepatocellular carcinoma and is currently treated with pegylated interferon‐α (peg‐IFN‐α) and ribavirin. Unfortunately, these limited treatment options often produce significant side effects, and currently, complete eradication of virus with combined drug modalities has not yet been achieved for the majority of chronically HCV‐infected individuals. Restricted treatment options, lack of a universal cure for HCV and the link between chronic infection, liver cirrhosis and hepatocellular carcinoma necessitate design of novel drugs and treatment options. Understanding the relationship between the immune response, viral clearance and inhibition of viral replication with pharmacology‐based design can ultimately allow for complete eradication of HCV. This review focuses upon significant novel preclinical and clinical specifically targeted antiviral therapy (STAT‐C) drugs under development, highlights their mechanism of action, and discusses their impact on systemic viral loads and permanent clearance of infection.

Raltegravir Is a Potent Inhibitor of XMRV, a Virus Implicated in Prostate Cancer and Chronic Fatigue Syndrome

Background Xenotropic murine leukemia-related retrovirus (XMRV) is a recently discovered retrovirus that has been linked to human prostate cancer and chronic fatigue syndrome (CFS). Both diseases affect a large fraction of the world population, with prostate cancer affecting one in six men, and CFS affecting an estimated 0.4 to 1% of the population. Principal Findings Forty-five compounds, including twenty-eight drugs approved for use in humans, were evaluated against XMRV replication in vitro. We found that the retroviral integrase inhibitor, raltegravir, was potent and selective against XMRV at submicromolar concentrations, in MCF-7 and LNCaP cells, a breast cancer and prostate cancer cell line, respectively. Another integrase inhibitor, L-000870812, and two nucleoside reverse transcriptase inhibitors, zidovudine (ZDV), and tenofovir disoproxil fumarate (TDF) also inhibited XMRV replication. When combined, these drugs displayed mostly synergistic effects against this virus, suggesting that combination therapy may delay or prevent the selection of resistant viruses. Conclusions If XMRV proves to be a causal factor in prostate cancer or CFS, these discoveries may allow for rational design of clinical trials.

Approaches to hepatitis C treatment and cure using NS5A inhibitors.

Recent progress in the understanding of hepatitis C virus (HCV) biology and the availability of in vitro models to study its replication have facilitated the development of direct-acting antiviral agents (DAAs) that target specific steps in the viral replication cycle. Currently, there are three major classes of DAA in clinical development: NS3/4A protease inhibitors, NS5B polymerase inhibitors, and NS5A directed inhibitors. Several compounds thought to bind directly with NS5A are now in various clinical trial phases, including the most advanced, daclatasvir (BMS-790052), ledipasvir (GS-5885), and ABT-267. While many NS5A-targeted compounds demonstrate picomolar potency, the exact mechanism(s) of their action is still unclear. In the clinic, NS5A HCV inhibitors show promise as important components in DAA regimens and have multifunctionality. In addition to inhibiting viral replication, they may synergize with other DAAs, possibly by modulating different viral proteins, to help suppress the emergence of resistant viruses. Structure-based models have identified target interaction domains and spatial interactions that explain drug resistance for mutations at specific positions (eg, residues 93 and 31) within NS5A and potential binding partners. This review provides, insights into the unique complexity of NS5A as a central platform for multiple viral/host protein interactions, and possible mechanism(s) for the NS5A inhibitors currently undergoing clinical trials that target this nonstructural viral protein.

Zika in the Americas, year 2: What have we learned? What gaps remain? A report from the Global Virus Network

In response to the outbreak of Zika virus (ZIKV) infection in the Western Hemisphere and the recognition of a causal association with fetal malformations, the Global Virus Network (GVN) assembled an international taskforce of virologists to promote basic research, recommend public health measures and encourage the rapid development of vaccines, antiviral therapies and new diagnostic tests. In this article, taskforce members and other experts review what has been learned about ZIKV-induced disease in humans, its modes of transmission and the cause and nature of associated congenital manifestations. After describing the make-up of the taskforce, we summarize the emergence of ZIKV in the Americas, Africa and Asia, its spread by mosquitoes, and current control measures. We then review the spectrum of primary ZIKV-induced disease in adults and children, sites of persistent infection and sexual transmission, then examine what has been learned about maternal-fetal transmission and the congenital Zika syndrome, including knowledge obtained from studies in laboratory animals. Subsequent sections focus on vaccine development, antiviral therapeutics and new diagnostic tests. After reviewing current understanding of the mechanisms of emergence of Zika virus, we consider the likely future of the pandemic.

Efficacy and safety of 3-week response-guided triple direct-acting antiviral therapy for chronic hepatitis C infection: a phase 2, open-label, proof-of-concept study.

BACKGROUND To shorten the course of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, we examined the antiviral efficacy and safety of 3 weeks of response-guided therapy with an NS3 protease inhibitor and dual NS5A inhibitor-NS5B nucleotide analogue. METHODS In this open-label, phase 2a, single centre study, Chinese patients with chronic HCV genotype 1b infection without cirrhosis were randomly allocated by a computer program to one of three treatment groups (sofosbuvir, ledipasvir, and asunaprevir; sofosbuvir, daclatasvir, and simeprevir; or sofosbuvir, daclatasvir, and asunaprevir) until six patients in each group (1:1:1) achieved an ultrarapid virological response (plasma HCV RNA <500 IU/mL by day 2, measured by COBAS TaqMan HCV test, version 2.0). Patients with an ultrarapid virological response received 3 weeks of therapy. Patients who did not achieve an ultrarapid response were switched to sofosbuvir and ledipasvir for either 8 weeks or 12 weeks. The primary endpoint was the proportion of patients with a sustained virological response at 12 weeks (SVR12) after treatment completion, analysed in the intention-to-treat population. All patients who achieved an ultrarapid virological response were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT02470858. FINDINGS Between April 5, 2015, and April 15, 2015, 26 eligible patients were recruited. 12 patients were assigned to sofosbuvir, ledipasvir, and asunaprevir; six to sofosbuvir, daclatasvir, and simeprevir; and eight to sofosbuvir, daclatasvir, and asunaprevir. Six patients in each group achieved an ultrarapid virological response (18 [69%]). All patients with an ultrarapid virological response who were given 3 weeks of triple therapy achieved SVR12. The most common adverse events were fatigue (one [17%] of six patients receiving sofosbuvir, ledipasvir, and asunaprevir; one [17%] of six patients receiving sofosbuvir, daclatasvir, and simeprevir; and two [33%] of six patients receiving sofosbuvir, daclatasvir, and asunaprevir) and headache (one [17%] patient in each group). No patients experienced any serious adverse events. INTERPRETATION In this proof-of-concept study, all patients with chronic HCV without cirrhosis who achieved an ultrarapid virological response on triple direct-acting antiviral regimens by day 2 and received 3 weeks of treatment were cured, with excellent tolerability. By shortening the duration of therapy from the currently recommended 12 weeks to 3 weeks, we could drastically reduce the cost of therapy and the rate of adverse events. Further large-scale studies should be done to confirm our findings. FUNDING Center for AIDS Research, National Institutes of Health, US Department of Energy, National Center for Research Resources and the Office of Research Infrastructure Programs, Cheng Si-Yuan (China-International) Hepatitis Research Foundation, and Humanity and Health Medical Group.

Hepatitis G Virus: Prevalence and Sequence Analysis in Blood Donors of São Paulo, Brazil

Background and objectives: Hepatitis G virus (HGV) is a recently discovered viral agent transmitted by blood, which was firstly identified in patients with acute or chronic liver disease. HGV prevalence in US blood donors was recently found to average 1–2%. We report a much higher HGV frequency among blood donors of São Paulo, Brazil. Materials and methods: 200 serum samples were submitted to RT-PCR using primers directed to the 5′ untranslated region and nonstructural 5A (NS5A) region. PCR products were analyzed by gel electrophoresis and Southern blot hybridization. Results: Of the 200 specimens, 18 (9%; 95% CI 5.4–13.8%) were positive by both sets of primers. Sequence analysis of the NS5A PCR products revealed a homology of 96.3%. Of the 18 HGV-positive symples, only one was positive for anti-HBc and all were anti-HCV- and HCV-RNA-negative. Conclusion: Such a high prevalence of HGV in a nonsymptomatic population suggests that this is a benign agent.

Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1.

ABSTRACT In order to survive prolonged treatment with antiretroviral nucleoside analogs, the human immunodeficiency virus type 1 (HIV-1) is selectively forced to acquire mutations in the reverse transcriptase (RT) gene. Some of these mutations are more common than others and have become markers for antiretroviral resistance. For the early detection of these markers, a novel MultiCode-RTx one-step testing system to rapidly and simultaneously characterize mixtures of HIV-1 targets was designed. For cDNA, nucleotide polymorphisms for codon M184V (ATG to GTG) and K65R (AAA to AGA) could be differentiated and quantified even when the population mixture varied as much as 1 to 10,000. Standard mixed-population curves using 1 to 100% of the mutant or wild type generated over 4 logs of total viral particle input did not affect the overall curves, making the method robust. The system was also applied to a small set of samples extracted from infected individuals on nucleoside reverse transcriptase inhibitor therapy. Of 13 samples tested, all were positive for HIV and 10 of the 13 genotypes determined were concordant with the line probe assay. MultiCode-RTx could be applied to other drug-selected mutations in the viral genome or for applications where single-base changes in DNA or RNA occur at frequencies reaching 0.01% to 1%, respectively.

Synthesis and anti-hepatitis B virus and anti-hepatitis C virus activities of 7-deazaneplanocin A analogues in vitro.

A series of 7-deazaneplanocin A (7-DNPA, 2) analogues were synthesized and evaluated for in vitro antiviral activity against HBV and HCV. The syntheses of target carbocyclic nucleosides were accomplished via a convergent procedure. 7-Substitutions were introduced by using 7-substituted-7-deaza heterocyclic base precursors (F, Cl, Br, and I) or via substitution reactions after the synthesis of the carbocyclic nucleosides. Among the synthesized compounds, 2, 13-15, 24, and 27 exhibited significant anti-HCV activity (EC(50) ranged from 1.8 to 20.1 microM) and compounds 2, 15, 22, and 24 demonstrated moderate to potent anti-HBV activity (EC(50) = 0.3-3.3 microM). In addition, compound 24 also showed activity against lamivudine- and adefovir-associated HBV mutants.