We have recovered five infectious molecular clones of the lentivirus equine infectious anaemia virus (EIAV). The clones were recovered from fetal equine kidney (FEK) cells infected with a virulent, cell culture-adapted virus stock (designated PV) and have been characterized at a molecular level. Each clone has unique envelope and long terminal repeat (LTR) sequences. We further investigated LTR sequence variation in the PV stock using PCR amplification to obtain additional LTR clones from infected FEK cells and from peripheral blood mononuclear ceils (PBMCs) from animals experimentally infected with PV.

To examine the relationship between macrophage tropism and neurovirulence, macaques were inoculated with two recombinant hybrid viruses derived from the parent viruses SIVmac239, a lymphocyte-tropic, non-neurovirulent clone, and SIV/17E-Br, a macrophage-tropic, neurovirulent virus strain. The first recombinant, SIV/17E-Cl, contained the portion of the env gene that encodes the surface glycoprotein and a short segment of the transmembrane glycoprotein of SIV/17E-Br in the backbone of SIVmac239. Unlike SIVmac239, SIV/17E-Cl replicated productively in macrophages, demonstrating that sequences in the surface portion of env determine macrophage tropism. None of five macaques inoculated with SIV/17E-Cl developed simian immunodeficiency virus (SIV) encephalitis. The second recombinant, SIV/17E-Fr, which contained the entire env and nef genes and the 3' long terminal repeat of SIV/17E-Br in the SIVmac239 backbone, was also macrophage tropic. Six of nine macaques inoculated with SIV/17E-Fr developed SIV encephalitis ranging from mild to moderate in severity, indicating a significant (P = 0.031) difference in the neurovirulence of the two recombinants. In both groups of macaques, CD4+ cell counts declined gradually during infection and there was no significant difference in the rate of the decline between the two groups of macaques. This study demonstrated that macrophage tropism alone is not sufficient for the development of neurological disease. In addition, it showed that while sequences in the surface portion of the envelope gene determine macrophage tropism, additional sequences derived from the transmembrane portion of envelope and/or nef confer neurovirulence.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibodycoated platelets from the peripheral circulation by hepatic Kupifer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication. * Corresponding author. and for platelet-associated virus. We have also examined bone marrow for the presence of viral replication in megakaryocytes or megakaryocyte depletion.

To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3 end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo.