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During the epidemic caused by maedi-visna virus (MVV) of sheep in Iceland, the pulmonary affection, maedi, was the predominant clinical manifestation. In some flocks, however, a central nervous system (CNS) affection, visna, was the main cause of morbidity and mortality. As there is only one breed of sheep in the country, host factors did apparently not play an important role in the different clinical manifestations. To obtain some information on possible viral genetic determinants of neurotropism and neurovirulence we studied both phenotypic and genotypic properties of two maedi-visna virus strains; a strain that was originally isolated from the brain of sheep with encephalitis (visna), and another strain isolated from the lungs of a sheep suffering from pneumonia (maedi). The brain isolate was found to grow faster in sheep choroid plexus cells than the lung isolate, whereas the growth rate in macrophages was similar for the maedi and visna virus strains. Intracerebral inoculation indicated that the visna virus isolate induced more severe brain lesions than the maedi isolate. In addition, a pathogenic molecular clone derived from a visna strain (KV1772kv72/67) was tested for growth in sheep choroid plexus cells and macrophages. The molecularly cloned virus retained the fast growth rate in choroid plexus cells. The nucleotide sequence of the env gene and the U3 of the LTR was determined for the maedi strain and compared to that of the visna strains. There was an 11.7% difference in deduced amino acid sequence in the Env protein and a 6% difference in the LTR. The molecular clone KV1772kv72/67 will be a useful reagent for characterization of viral determinants of cell tropism in vitro and possibly neurovirulence in vivo.

Intracerebral serial passage of visna virus KV1514 through three icelandic sheep was used to select for strains with increased neurovirulence. A strain (KV1772) with increased neuropathogenicity was obtained. We isolated several proviral molecular clones from a plaque-purified biological clone of KV1772 that induced typical visna virus pathology in young sheep. One of the clones (kv72) was infectious, while others contained mutations or were permuted and required gene recombination with other proviral clones to generate infectious virus after transfection. Stable plasmids containing functional, full-length, visna virus KV1772 genomes were constructed from the proviral molecular clones. The in vitro cytopathic effects of virus derived from these clones varied depending upon the tissue origin of the infected cells. A goat cell line became persistently infected with molecularly cloned KV1772 virus; these cells resisted the cell-killing effects and continuously shed high levels of infectious virus. We determined the complete nucleotide sequence of a KV1772 provirus; it contains open reading frames for all structural and accessory genes previously identified in the visna virus genome and is highly homologous to other published visna virus sequences. Progeny of molecularly cloned KV1772 virus rapidly induced both a pronounced neuropathology and an unexpected, strong, neutralizing antibody response in experimentally infected young Icelandic sheep. The availability of stable plasmids of replication-competent and pathogenic proviral molecular clones of visna virus should now enable the study of the genetic determinants of neurovirulence and their interaction with the host immune system in visna virus pathogenesis.