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The number of dengue virus infections is increasing and the dengue NS2B–NS3 protease is considered a promising target for the development of antiviral therapies. Therefore, reliable and fast screening systems are needed for the discovery of new lead structures. In this chapter, we descri ...

Large-scale antiviral drug discovery and systematic identification of host factors promoting or restricting virus replication require robust, scalable, and cost-effective assay systems that allow for high throughput and automation. Here we describe the construction and ap ...

We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four d ...

Dengue, a mosquito-borne virus of the Flaviviridae family, is reemerging as one of the most important human pathogens in tropical and subtropical regions of the world. It is estimated that 2.5 billion people live in areas at risk for transmission of dengue virus (DENV). Furthermore, it causes sign ...

A hepatitis C virus (HCV) replicon-based protease phenotyping assay has been developed that allows determining the susceptibility of a patient’s HCV protease sequence to HCV protease inhibitors. In brief, HCV protease sequences amplified from clinical samples are cloned in a transi ...

The current standard of care (SOC) for hepatitis C virus (HCV) genotype 1-infected patients consists of telaprevir or boceprevir in addition to pegylated interferon and ribavirin treatment. Other selective inhibitors of HCV replication are being developed. Drug pressure may, depen ...

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase is essential for viral replication and a clinically validated antiviral target. Discovery of HCV polymerase inhibitors is greatly facilitated by the availability of a suitable biochemical assay using purified NS5B. We de ...

Kinetic profiling of drug binding to its target reveals important mechanistic parameters including drug–target residence time. In this chapter, we focus on global progress curve analysis as a convenient method for kinetic profiling. Detailed guidelines with pros and cons for various ...

This chapter describes the procedures for the generation of recombinant hepatitis B virus (HBV) that contains the polymerase/reverse transcriptase (pol/RT) gene from clinical isolates. It also contains procedures for phenotypic testing of the recombinant HBV in HepG2 cells for su ...

Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infec ...

Genotypic testing based on subtype-specific amplification and population Sanger sequencing for two nonstructural (NS) protein-coding regions, the NS3/4A protease and the NS5B polymerase, of the hepatitis C virus (HCV) genome is described here. The protocols include the molecular ...

Surface plasmon resonance (SPR)-based optical biosensors have been widely used to study biomolecular interactions, and applied to many areas of drug discovery including target identification, fragment screening, lead compound selection, early ADME (absorption, distribu ...

This chapter describes the procedures for production of recombinant hepatitis C virus (HCV) NS3 protease from clinical samples, which can be used in the biochemical assays to assess the impact of different drug-resistant mutations in the NS3 protein from patients under therapy of protea ...

Major advances in antiretroviral (ARV) therapy during the last decade have made HIV-1 infections a chronic, manageable disease. In spite of these significant advancements, ARV drug resistance remains a hurdle for HIV-infected patients who are committed to lifelong treatments. Seve ...

Human immunodeficiency virus type 1 (HIV-1) integrase is, in addition to reverse transcriptase and protease, an important enzymatic target for antiretroviral drug development. Integrase plays a critical role in the HIV-1 life cycle coordinating the integration of the reverse-tra ...

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) converts the viral single-stranded RNA into double-stranded DNA. The inhibition of reverse transcription in the viral life cycle has proven its efficacy as a clinically relevant antiviral target, but the appe ...

The human immunodeficiency virus type 1 (HIV-1) initiates infection through sequential interactions with CD4 and chemokine coreceptors unmasking the gp41 subunit of the viral envelope protein. Consequently, the N-terminal heptad repeats of gp41 form a trimeric coiled-coil groo ...

Highly active antiretroviral therapy (HAART) dramatically increases the long-term survival rates of human immunodeficiency virus type 1 (HIV-1) infected patients. Yet, poor adherence to therapy, adverse effects and the occurrence of resistant viruses can compromise the effic ...

Interactions among neurotoxicants in in vitro models, where the molecular mechanisms of toxicity are generally studied, represent today an emerging field in the experimental neurotoxicology. In this chapter, we define some general concepts about the optimization of in vitro exper ...

Cholesterol homeostasis is highly regulated in the nervous system; dysregulation in cholesterol trafficking and content have been involved in the pathogenesis of neurodegenerative diseases (such as Parkinson’s and Alzheimer’s diseases). Furthermore, low cholesterol lev ...