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The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Nucleotide sequence analyses of the env genes of two neurotropic variants of SIV mac 239 were performed to determine whether molecular changes in these genes could be correlated with neurotropism. Biological characterization of virus from the infectious molecular clone of SIV mac 239 had shown that it is highly lymphocyte-tropic and poorly macrophage-tropic. This virus failed to replicate in the brain after intracerebral inoculation, but passage of this virus in macaques resulted in development of vital variants that had acquired cell tropism for macrophages and were neurovirulent (D. P. Sharma, M. C. Zink, H. Anderson, R. J. Adams, J. E. Clements, S. V. Joag, and O. Narayan, J. Virol. , 66, 3550-3556, 1992). The neurotropic virus SIV mac 239/R71 was obtained from the brain of a monkey after the third in vivo passage of SIV mac 239. Inoculation of this virus into another macaque leads to CNS disease and the isolation of another neurotropic virus SIV mac 239/17E. The viral env sequences obtained by polymerase chain reaction amplification directly from DNA obtained from the brain of R71 and 17E macaques had a limited number of changes dispersed throughout the env gene when compared to the parental virus, SIV mac 239. The most important finding was that there was a common set of nucleotide changes in the env gene of both R71 and 17E. This suggested that viruses containing these changes had a selective growth advantage in the brain and were the predominant species present in the central nervous system of macaques R71 and 17E. Analysis of individual clones containing the R71 env gene revealed that different env genes were present, but all had the changes that were conserved in both R71 and 17E but not present in the original lymphocyte-tropic parental virus, SIV mac 239. Construction of an infectious recombinant virus containing the tat, rev , and env genes from 17E and the remainder of the genome from the parental virus SIV mac 239 resulted in a virus that had the macrophage-tropism of 17E virus isolated from brain. This demonstrates that the env gene of 17E confers the cellular tropism of the virus on the parental virus, SIV mac 239.