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Molecular Mechanisms of Viral Attenuation

M. Manchester, D. Eto

Studies defining viral tropism, replication, and pathogenesis typically use laboratory-passaged strains rather than primary isolates from patients. Most studies on measles virus have been done with such laboratory strains. Surprisingly, although differences between vaccine strains and primary isolates from patients have been defined at the nucleotide level for measles virus, little is known about the mechanisms by which wild-type strains of the virus cause acute disease, whereas the laboratory or vaccine strains do not. Such research is important not only for studies on measles virus but also for any system in which laboratory-passaged isolates are used to define the pathogenic properties of a virus.

We are studying use of cell-surface receptors and tissue-culture adaptation by strains of wild-type measles virus isolated from patients. The major cellular receptor for measles virus, CD46, is a regulatory protein for complement and was detected by using 2 laboratory passaged strains. Adaptation of isolates of wild-type measles virus to tissue culture leads to profound differences in tropism and receptor usage. The phenotypes of the selected viruses depend on the cell type used for the selection. The ability of wild-type isolates to use CD46 for entry was examined either before or after their adaptation to tissue culture.

Isolates of wild-type measles virus obtained from and passaged solely on primary human peripheral blood mononuclear cells were lymphotropic and used human CD46 as a receptor to enter lymphoid cells. Surprisingly, once adapted to a widely used marmoset B-lymphocyte cell line, the wild-type isolates were no longer inhibited by the same anti-CD46 antibody. These findings indicate that culture on this cell line selects for viruses that have gained the ability to use an additional, alternative receptor not used by the initial isolate. Although historically measles virus has been considered a genetically stable virus, these results highlight major phenotypic differences between wild-type isolates passaged in peripheral blood mononuclear cells and isolates adapted to growth in cell lines and indicate that laboratory culture of wild-type measles virus can profoundly alter the phenotypes of the isolates.

Unlike the laboratory strain, lymphotropic wild-type strains of measles virus isolated from patients did not complete a full round of replication or produce infectious virus in CD46-expressing fibroblasts. Nevertheless, early transcription of wild-type measles virus mRNAs was detected in CD46-expressing fibroblasts, a finding that indicates that viral entry did occur in these cells. These results suggest that the tropism of wild-type viruses is determined not only by CD46 but also by other cellular cofactors at a step that occurs after entry. These findings highlight major phenotypic differences between attenuated viruses and the wild-type counterparts of the viruses and suggest that the altered tropism seen with laboratory viruses in culture may be a mechanism for attenuation and reduced pathogenicity in vivo.