Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 99, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/52610
Keywords
Immunology; Issue 99; HIV-1; Recombinant; Mutagenesis; Viral replication fitness; Growth competition; Fitness calculation
Categories
Funding
- US Public Health Services [P01AI057005, R01AI047734, R01AI111806]
- Functional Profiling and Computational Biology Core of the University of Washington's Center for AIDS Research [P30 AI027757]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available