Isolation and Characterization of Equine Infectious Anemia Virus Bacteriophage Lambda Clones

Abstract

Proviral cloning of EIAV was attempted from two unique cell lines. The cell line designated clone-14 was a clonal cell line which contained 6 integrated proviruses. The second cell line, designated clone-0.5, was the result of a de novo infection followed by single cell cloning and contained only 2 proviruses. Both of these cell lines produced infectious virus. Analysis of clones from the clone-14 cell line detected no apparent restriction fragment polymorphisms in 10/18 clones compared to a previously isolated and characterized, though noninfectious, proviral clone, clone-12. Sixteen of the analyzed clones were transfected into canine cell lines and produced EIAV p26 upon immunofluorescent observation. As detected by immunoblotting, six produced low levels of p26 in the culture medium, compared to the cell line from which they were cloned. Exposure of uninfected Cf2Th cells to this culture medium did not cause a productive infection. Only two populations of EcoRI fragments were expected from the clone-0.5 library. However, five different sizes were isolated. Six clones were analyzed and three were positive by immunofluorescence and immunoblot for p26. The apparent inability to isolate an infectious clone, even after enriching for proviral sequences and screening an excessive number of proviral genomes, was surprising. This is a common problem among lentiviruses as a literature search revealed.