Electronic Thesis and Dissertation Repository

Thesis Format



Master of Science


Microbiology and Immunology


Troyer, Ryan

2nd Supervisor

Arts, Eric



This project sought to perform the in vitro work needed to accomplish the long-term vision of harnessing the similarities between HIV (Human Immunodeficiency Virus) and FIV (Feline Immunodeficiency Virus) to develop an animal model whereby cats can be used to study HIV pathogenesis and therapeutics. We transfected CRFK (Crandell Rees Feline Kidney) fibroblasts with plasmids that could express human or feline CD4, CCR5, or both, and determined receptor surface expression through flow cytometry. We discovered that HIV envelope expressed on 293T can fuse with huCD4/huCCR5 on CRFK. These cat cell lines were also capable of supporting HIV infection. Additionally, we evaluated whether the yeast recombination system could be used to clone FHIVenv chimeras wherein the HIV env is inserted into the FIV backbone in place of FIV env. In the future, FHIVenv adapted to replicate in the cat cell lines produced herein, can be tested in in vivo in cats.

Summary for Lay Audience

The lack of suitable, cost-effective animal models remains a challenge in studying HIV (Human Immunodeficiency Virus). Cats are natural hosts for FIV (Feline Immunodeficiency Virus), which is similar in structure and pathogenesis to HIV. This project’s goal was to establish the in vitro work needed to accomplish the long-term vision of utilizing cats in vivo to study HIV therapeutics and vaccines. The in vitro work involves creating chimeric viruses as well as engineering the cell lines required for testing viral envelope interactions with entry receptors. These viruses and cell lines are also required for the infectivity assays. We sought to execute this through the creation of FHIVenv chimeras composed of HIV envelope genes inserted into the FIV genome, in place of FIV envelope. We attempted several DNA cloning strategies to accomplish this. We also engineered CRFK (Crandell Rees Feline Kidney) Fibroblast cell lines to express human and feline CD4 and CCR5 and tested the receptor expression level on their surface. We were able to achieve higher human CD4/CCR5 expression than feline CD4/CCR5 expression on independent CRFK cell lines. Human CD4 and CCR5 are typically used by HIV to enter and infect human cells. We found that not only can HIV envelope proteins from diverse HIV subtypes and strains bind to human CD4 and CCR5 expressed on cat cells, HIV-AD8 (a subtype B virus) can also use these receptors to enter and infect cat cells. In the future, receptor-envelope interactions can be tested between these diverse HIV envelopes and the engineered feline CD4/CCR5 CRFK. The envelopes that are capable of interacting with these feline receptors can be cloned into FHIVenvs, following which infectivity assays can be conducted, where the chimeras can be used to infect feCD4/feCCR5 CRFK. The eventual goal is to test these chimeras in vivo in cats. If they are pathogenic, infectious, and replication-competent in vivo, cats can be utilized as model organisms to develop and test therapeutics and vaccine strategies targeting the HIV envelope.