Transspecies transmission of the endogenous koala retrovirus

Abstract

The koala retrovirus (KoRV) is a gammaretrovirus closely related to the gibbon ape leukemia virus and induces leukemias and immune deficiencies associated with opportunistic infections, such as chlamydiosis. Here we characterize a KoRV newly isolated from an animal in a German zoo and show infection of human and rat cell lines in vitro and of rats in vivo, using immunological and PCR methods for virus detection. The KoRV transmembrane envelope protein (p15E) was cloned and expressed, and p15E-specific neutralizing antibodies able to prevent virus infection in vitro were developed. Finally, evidence for immunosuppressive properties of the KoRV was obtained.

FIG. 1.

Infection studies with KoRV. (a) Detection of provirus in different cell lines before and after infection with KoRV using PCR. PBMCs from a healthy koala were taken as the positive control. (b) Fifty percent tissue culture infective dose (TCID50) values (means and standard deviations) of KoRV produced from different cell lines and titrated on human 293 cells. (c) Transmission electron microscopy of KoRV produced by human 293 kidney cells and (d) KoRV particles in koala lymphoma cells.

FIG. 2.

Biochemical and immunological characterization of KoRV. (a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose gradient-purified KoRV produced by human 293 cells with Coomassie blue staining. (b) Western blot analysis of the same material using a rat antiserum specific for KoRV p15E that recognized p15E and the precursor gp85. (c) Western blot analysis of cell lysates from human 293 cells producing KoRV from (lane 1) human 293 cells producing PERV and (lane 2) mouse NIH 3T3 cells producing MuLV and (lane 3) using a goat antiserum specific for PERV p27Gag. (d) Western blot analyses using recombinant p15E of KoRV as antigens and sera from three different rats (A, B, C) immunized with recombinant p15E (rp15E) of KoRV. (e) Neutralizing activity of rat sera (immune sera [IS]) in comparison to that of preimmune sera (PS) at a dilution of 1:8. Infection was measured as the percentage of provirus integration using real-time PCR. Results are expressed as means and standard deviations.

FIG. 3.

Influence of purified KoRV and mock preparations on IL-10 production. Supernatants from 293 cells producing KoRV and from uninfected 293 cells (mock) were ultracentrifuged, and pellets were incubated overnight at different concentrations with human PBMCs from healthy donors. IL-10 was measured in the supernatant using an enzyme-linked immunosorbent assay, and results are expressed as means (and standard deviations) after subtracting the concentration of IL-10 released by untreated cells.