Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses
| Autor: | J. Victor Swain, Brandon S. Maust, J. Sunshine, Jan McClure, Siriphan Manocheewa, Yi Liu, Moon S. Kim, Erinn Lanxon-Cookson, Morgane Rolland, Ushnal Rao, Wenjie Deng, James I. Mullins |
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| Rok vydání: | 2015 |
| Předmět: |
DNA Replication
Issue 99 media_common.quotation_subject General Chemical Engineering Immunology HIV Core Protein p24 Human immunodeficiency virus (HIV) Clone (cell biology) Biology Virus Replication medicine.disease_cause Competition (biology) General Biochemistry Genetics and Molecular Biology Multiplicity of infection Replication (statistics) medicine Humans media_common Protocol (science) Genetics Recombinant General Immunology and Microbiology General Neuroscience 3. Good health Viral replication fitness HEK293 Cells Viral replication Mutagenesis DNA Viral Mutation HIV-1 Fitness calculation Growth competition Pairwise comparison |
| Zdroj: | Journal of Visualized Experiments : JoVE |
| ISSN: | 1940-087X |
| Popis: | In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories. |
| Databáze: | OpenAIRE |
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