RESUMO
We have genetically engineered a panel of recombinant measles viruses (rMVs) that express from various positions within the MV genome either the HN or F surface glycoproteins of mumps virus (MuV) or the env, gag or pol proteins from simian immunodeficiency virus (SIV). All rMVs were rescued from the respective antigenomic plasmid constructs; progeny viruses replicated comparably to the progenitor Edmonston B MV, but showed slight propagation retardation, which was dependent on the size and nature of the expressed proteins and on the genomic position of the inserts. All transgenes except that encoding mumps F glycoprotein were faithfully maintained and expressed even after virus amplification by 10(20). Our results suggest possible applications of rMVs as live-attenuated, multivalent vaccines against retroviruses such as SIV and HIV as well as other pathogens more distantly related to MV than MuV.
Assuntos
Antígenos Virais/genética , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Vírus da Caxumba/genética , Vírus da Caxumba/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Animais , Sequência de Bases , Chlorocebus aethiops , Primers do DNA/genética , Expressão Gênica , Genes Virais , Engenharia Genética , Vetores Genéticos , Vírus do Sarampo/crescimento & desenvolvimento , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Recombinação Genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação , Células Vero , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
