Targeted ablation of exon 2 of the Avian Leukosis Virus-A (ALV-A)receptor gene in a chicken fibroblast cell line by CRISPR abrogates ALV-A infection

Authors: Conrad, Steven; Mays, Jody; Hearn, Cari; Dunn, John

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2022
Publication Date: 2/21/2023
Citation: Conrad, S.J., Mays, J.K., Hearn, C.J., Dunn, J.R. 2023. Targeted ablation of exon 2 of the Avian Leukosis Virus-A (ALV-A)receptor gene in a chicken fibroblast cell line by CRISPR abrogates ALV-A infection. Avian Diseases. 67(1):102-107. https://doi.org/10.1637/aviandiseases-D-22-00072.
DOI: https://doi.org/10.1637/aviandiseases-D-22-00072

Interpretive Summary: The avian leukosis viruses (ALVs) are a related family of highly-contagious retroviruses which infect chickens and turkeys, and can cause serious problems for poultry producers. As part of its mission, the Avian Diseases and Oncology Laboratory (ADOL) accepts samples from field isolates of clinically-ill chickens and characterizes the infectious organisms. For the ALVs ADOL currently uses chickens who are genetically resistant to the various strains of ALVs as part of the diagnostic process. We wished to eliminate the expense and manpower burden of maintaining these chicken lines by creating cell lines which are individually unable to host particular strains of ALV, thereby providing us with diagnostic tools which are easy and inexpensive to maintain. To this end, we used gene editing technology to destroy the receptor the ALV-A subtype virus uses to enter chicken cells, thereby creating a cell line which is impervious to ALV-A infection. In this paper we characterize the deletions in the Tva gene and demonstrate that these cells are not able to host ALV-A infection.

Technical Abstract: The U.S. Department of Agriculture Avian Disease and Oncology Laboratory currently relies on live birds of specific genetic backgrounds for producing chicken-embryo fibroblasts that are used for the diagnosis and subtyping of field isolates associated with avian leukosis virus (ALV) outbreaks. As an alternative to maintaining live animals for this purpose, we are currently developing cell lines capable of achieving the same result by ablation of the entry receptors utilized by ALV strains. We used CRISPR-Cas9 on the cell fibroblast-derived cell line DF-1 to disrupt the tva gene, which encodes the receptor required for binding and entry of ALV-A into cells. We ultimately identified seven DF-1 clones that had biallelic and homozygous indels at the Cas9 target site, exon 2 of tva. When tested in vitro for their ability to host ALV-A, the five clones that had frameshift mutations that disrupted the Tva protein were unable to support ALV-A replication. This result clearly demonstrates that modified cell lines can be used as part of a battery of tests to determine ALV subtype for isolate characterization, thus eliminating the need for live birds.