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.Retroviruses have long been known to be capable of infecting new host species by transspecies transmission; human immunodeficiency viruses (HIV) types 1 and 2 are the products of such a transspecies transmission (12, 13). The koala retrovirus (KoRV) is an example of a recent transspecies transmission and endogenization, whereas the closely related gibbon ape leukemia virus (GaLV) remains exogenous in gibbons. Although both viruses are related to endogenous retroviruses of South East Asian mice (18), the transmission routes are still unknown. Koalas in Australia as well as in many zoos suffer from leukemia (1,14) and from infections such as chlamydiosis (2). Since Chlamydia infections are characteristically opportunistic infections commonly associated with retroviral infections, such as HIV and feline immunodeficiency virus (4,5,20), it is likely that KoRV, as with many other retroviruses, is able to induce immunosuppression.A new KoRV isolate was obtained from mitogen-stimulated peripheral blood mononuclear cells (PBMCs) of a healthy male animal from the Duisburg Zoo, Duisburg, Germany (designated KoRV Duisburg-Berlin [KoRV D-B ]). This strategy has been used previously to isolate porcine endogenous retroviruses (PERVs) from pig PBMCs (24). To study the host range of KoRV, human 293 kidney cells and the human T lymphocyte lines C8166 and CEM, as well as rat and mouse fibroblasts (rat1 and NIH 3T3, respectively), were used. Provirus integration was shown by PCR in all cell lines except NIH 3T3 (Fig. 1a), and the release of infectious virus was shown by the titration of cell-free supernatant on uninfected 293 (Fig. 1b) or C8166 cells. In addition, virus production was demonstrated by electron microscopy ( Fig. 1c and d). Whereas virus particles produced after three passages on human 293 cells were characterized by a uniform morphology, particles from a lymphoma from an animal at the Antwerp Zoo (Fig. 1d) showed pleomorphic particles as described previously for the cells of a leukemic koala (26).The entire env gene (p15E and gp70) was amplified from the DNA of the animal from which KoRV D-B was isolated and sequenced. The sequence revealed an almost perfect match (14). When the sequences of the ectodomains of p15E amplified from the DNA of one other healthy animal and one diseased animal were compared, no differences were found.Biochemical and immunological chara...

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particleassociated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, a goat antiserum against the recombinant ectodomain of the transmembrane envelope protein p15E was found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, E1 (GPQQLEK) and E2 (FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, antibodies isolated from AIDS patients and specific for sequences of HIV-1 partially homologous with E2 (Mab4E10, LWNWFN) or located in close proximity to E2 (Mab2F5, ELDKWA) are known to neutralize several strains of HIV-1. It is the first report showing epitope mapping of gammaretrovirus-specific neutralizing antibodies and demonstrating similarity to corresponding epitopes in HIV. These domains of the transmembrane proteins of different retroviruses are an effective target for neutralizing antibodies and may be a useful antigen to create an antiretroviral vaccine.

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising. To detect neutralising antibodies specific for PERV, a novel assay was developed, which is based on quantification of provirus integration by real-time PCR. In addition, for the first time, highly effective neutralising antibodies were obtained by immunisation with the surface envelope protein of PERV. These data indicate that neutralising antibodies can be induced by immunisation with both envelope proteins.

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