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Methods for assessing feline immunodeficiency virus infection, infectivity and purification
Melanie Ammersbach1, Dorothee Bienzle
1Department of Pathobiology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Insights
Feline immunodeficiency virus (FIV) infection in cats mirrors human immunodeficiency virus (HIV) infection. Purifying FIV is crucial for accurate viral quantification and understanding its effects on immune responses.
Area of Science:
- Virology
- Immunology
- Lentivirus Research
Background:
- Feline immunodeficiency virus (FIV) infection in cats shares similarities with human immunodeficiency virus (HIV) infection.
- Current diagnostic methods rely on antibody detection, but quantifying infectious viral particles requires advanced techniques.
- Existing assays cannot differentiate between infectious and defective viral particles.
Purpose of the Study:
- To review methods for purifying lentiviruses, specifically feline immunodeficiency virus (FIV).
- To evaluate the suitability of different purification techniques for FIV research.
- To highlight the impact of purification on immune responses and assays.
Main Methods:
- Review of lentivirus purification techniques including ultracentrifugation and chromatography.
- Discussion of methods for removing non-viral components like extraviral proteins and exosomes.
- Consideration of subtilisin digestion and immunoaffinity purification.
Main Results:
- Purification is essential for analyzing the viral proteome and host-virus interactions.
- Different purification methods offer varying advantages and disadvantages for FIV.
- Purification processes can influence downstream immune response analysis.
Conclusions:
- Accurate quantification of infectious FIV particles necessitates effective purification strategies.
- The choice of purification method impacts the integrity of viral particles and subsequent research findings.
- Understanding purification effects is vital for reliable FIV research and diagnostic assay development.
Abstract:
Infection of cats with the feline immunodeficiency virus (FIV) recapitulates many aspects of infection of humans with HIV, including highly activated but ineffectual immune responses. Infected hosts remain seropositive for life, and detection of antibodies is the mainstay of diagnosis. However, to quantify virus for research or prognosis, viral proteins, nucleic acids or enzymes, are typically measured by ELISA, PCR or activity, respectively. While such assays are in wide use, they do not distinguish whole, infectious viral particles from defective or disrupted viruses. Titers of infectious viral particles may be estimated from tissue culture infectious doses or by enumerating cell-associated viral proteins, viral transcriptional activity or formation of syncytia. To analyze the viral proteome and the incorporation of host components into viral envelopes, pure lentiviral preparations are required. Methods for purifying lentiviruses include ultracentrifugation to separate particles by size, mass and/or density; chromatography to separate particles by charge, affinity or size; and additional removal of extraviral proteins and exosomes through subtilisin digestion or immunoaffinity. This article reviews advantages and disadvantages of different approaches to purification of lentiviruses with special reference to suitability for FIV, and highlights effects of purification on immune responses and immune assays.
