By Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde (auth.), Edwin Yunhao Gong (eds.)
Although antiviral medicinal drugs were effectively built for a few viral illnesses, there is still a transparent, unmet clinical have to boost novel antiviral brokers for the regulate and administration of many viruses that at the moment haven't any or restricted cures in addition to a necessity to beat the constraints linked to the present antiviral medicinal drugs, resembling antagonistic results and emergence of drug-resistant mutations. the second one variation of Antiviral equipment and Protocols features:
- All chapters are new and written by means of specialists within the box, reflecting the key contemporary technical advances in antiviral learn and discovery.
- This variation specializes in many very important human viruses, akin to human immunodeficiency virus style 1 (HIV-1), hepatitis viruses (hepatitis B and C viruses), herpes viruses, human respiration syncytial virus (RSV), and influenza virus, whereas additionally that includes a few vital rising viruses, corresponding to dengue virus, West Nile virus, and chikungunya virus.
- As a quantity within the hugely profitable Methods in Molecular Biology sequence, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, without problems reproducible laboratory protocols, and pointers on troubleshooting and averting recognized pitfalls.
Comprehensive and state of the art, Antiviral tools and Protocols, moment Edition will function an exceptional laboratory reference for pharmaceutical and educational biologists, medicinal chemists, and pharmacologists in addition to for virologists within the box of antiviral learn and drug discovery.
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Extra resources for Antiviral Methods and Protocols
We use Efavirenz as a positive control, which is a known NNRTI. 2. 1, prepare a RT solution at 10× the final enzyme concentration in the assay by diluting the RT stock solution provided by the supplier. Typically, we dilute the RT stock solution 10,000 times in RPMI/10 % FBS. An SPA-Based HIV-1 Reverse Transcriptase Assay 23 3. Prepare the assay mix as described in Table 1 and keep on ice. 4. Add 25 μL of assay mix to all wells of the compound plate. The final volume in each well is 50 μL with a final DMSO concentration of 1 %.
First, integrase removes a dinucleotide from the 3′ viral DNA ends in a process called 3′-processing. Next, in a process called strand transfer, the viral DNA is integrated into the host genomic DNA. Early on, biochemical assays have played a critical role in understanding the function of HIV-1 integrase and the discovery of small-molecule inhibitors. In this chapter we describe two biochemical assays to identify inhibitors of the 3′-processing and strand transfer process of HIV-1 integrase. Key words HIV-1, Integrase, Strand transfer, 3′-Processing, Real-time PCR 1 Introduction The viral enzyme integrase (IN) is essential for the HIV replication cycle and is encoded by the pol gene.
The different steps of reverse transcription are well characterized and have been reviewed elsewhere . The RT incorporates deoxyribonucleotide triphosphates (dNTPs) at the 3′ end of the primer, using the viral RNA as a template. The inhibition of this step in the viral life cycle played a pivotal role in the establishment of highly active antiretroviral therapy as RT inhibitors are included in the current standard of care . Both nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) have proven their clinical efficacy.