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. 2008 Dec;82(23):11979-84.
doi: 10.1128/JVI.00867-08. Epub 2008 Sep 17.

Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

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Measles viruses possessing the polymerase protein genes of the Edmonston vaccine strain exhibit attenuated gene expression and growth in cultured cells and SLAM knock-in mice

Makoto Takeda et al. J Virol. 2008 Dec.

Abstract

Live attenuated vaccines against measles have been developed through adaptation of clinical isolates of measles virus (MV) in various cultured cells. Analyses using recombinant MVs with chimeric genomes between wild-type and Edmonston vaccine strains indicated that viruses possessing the polymerase protein genes of the Edmonston strain exhibited attenuated viral gene expression and growth in cultured cells as well as in mice expressing an MV receptor, signaling lymphocyte activation molecule, regardless of whether the virus genome had the wild-type or vaccine-type promoter sequence. These data demonstrate that the polymerase protein genes of the Edmonston strain contribute to its attenuated phenotype.

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Figures

FIG. 1.
FIG. 1.
Small plaque formed by the Edmonston strain and recombinant MVs with the P or L gene of the Edmonston strain on Vero/hSLAM cells. (A) Plaque assays were performed for various MV strains (three wild-type strains [IC323, JPN/50.98/1, and JPN/26.99/1] and two Edmonston lineage strains [Ed-ATCC and Ed-tag]). Monolayers of Vero/hSLAM cells on 12-well cluster plates were infected with 50 PFU of each virus and overlaid with Dulbecco's modified Eagle medium containing 2% fetal bovine serum and 1% methylcellulose. At 5 days p.i., the cells were stained with RTU Vectastain Elite ABC reagent (Vector Laboratories) using anti-MV H-protein monoclonal antibodies and a biotinylated secondary antibody. After high-resolution digital images were obtained, the sizes of all plaques were measured. The mean sizes ± standard deviations are shown in the bar graph. (B) Plaque assays were performed for wild-type IC323-EGFP and six recombinant MVs (each containing one of the Ed-tag strain genes in the backbone of the IC323-EGFP genome) on Vero/hSLAM cells, as described for panel A.
FIG. 2.
FIG. 2.
Attenuated gene expression by recombinant MVs with the P and/or L genes of the Edmonston strain. (A) Confluent monolayers of various cell lines (Vero/hSLAM, CV1/hSLAM, HeLa/hSLAM, CHO/hSLAM, and B95a) and suspensions of nonadherent MT2 cells cultured in 24-well cluster plates were infected with 2.5 × 103 PFU of IC323-Luci (circles), IC/Ed-P-Luci (squares), IC/Ed-L-Luci (triangles), and IC/Ed-PL-Luci (diamonds). After various intervals, the Renilla luciferase activities were measured. Data are means ± standard deviations for triplicate samples. RLU, relative light units. (B) Confluent monolayers of Vero/hSLAM cells in 6-cm culture plates were infected with 1.0 × 104 PFU of IC323-Luci (light gray bars), IC/Ed-P-Luci (black bars), IC/Ed-L-Luci (dark gray bars), and IC/Ed-PL-Luci (white bars) and cultured in the presence of a fusion-blocking peptide. At 18 h p.i., mRNAs were purified from the cells, and the levels of N, P, M, F, H, and L mRNAs were determined by reverse transcription-quantitative PCR. Data are means ± standard deviations for triplicate samples. (C) Minigenome assays. The method was described in detail elsewhere (19). Monolayers of CHO/hSLAM cells cultured in Opti-MEM on 24-well plates were infected with vTF7-3 at a multiplicity of infection of 0.5 and then transfected with 0.2 μg of p18MGFLuc01-wt-Le, 0.2 μg of pCAG-T7-IC-N, 0.3 μg of a P-protein expression plasmid (pCAG-T7-IC-PΔC or -Ed-PΔC), and 0.2 μg of an L-protein expression plasmid (pGEMCR-IC-L or -Ed-L) using Lipofectamine 2000 (Invitrogen). At 6 h p.i., the culture media were replaced with RPMI 1640 medium supplemented with 7.5% fetal bovine serum. At 48 h p.i., the firefly luciferase activities were measured. (-), L-protein expression plasmid was omitted. Data are means ± standard deviations for triplicate samples.
FIG. 3.
FIG. 3.
Neutral effects of substitutions in the leader region of the Edmonston strain on MV gene expression levels and plaque sizes. (A) Confluent monolayers of CV1/hSLAM, HeLa/hSLAM, and CHO/hSLAM cells cultured in 24-well cluster plates were infected with 2.5 × 103 PFU of IC323-Luci (circles) and IC/Ed-Le-Luci (triangles). After various intervals, the Renilla luciferase activities were measured. Data are means ± standard deviations for triplicate samples. RLU, relative light units. (B) Plaque assays were performed for the recombinant MVs, as described for Fig. 1A. The IC/Ed-Le-Luci, IC/Ed-LeP-Luci, IC/Ed-LeL-Luci, and IC/Ed-LePL-Luci genomes contain Ed-tag genome regions encoding Le alone, Le and P, Le and L, or Le, P, and L, respectively, in the backbone of the IC323-Luci genome.
FIG. 4.
FIG. 4.
Attenuated gene expression by the Edmonston strain in cultured cells and SLAM-knock-in mice. (A) Plaque assays were performed for IC323-Luci and Ed-tag-VH110Y/R272C-Luci on Vero/hSLAM cells, as described for Fig. 1A. (B) Confluent monolayers of HeLa/hSLAM, A549/hSLAM, CV1/hSLAM, B95a, and CHO/hSLAM cultured in 12-well cluster plates were infected with 2.5 × 103 PFU of IC323-Luci (black bars) and Ed-tag-VH110Y/R272C-Luci (gray bars). At 30 h p.i., the Renilla luciferase activities were measured. Data are means ± standard deviations for triplicate samples. Mock-infected samples are shown by white bars. RLU, relative light units. (C) IC323-Luci, IC/Ed-L-Luci, and Ed-tag-VH110Y/R272C-Luci (1.5 × 105 PFU) were injected into the peritoneal cavities of IFNAR1−/− SLAM-knock-in mice. At 5 days p.i., the spleens were removed and analyzed for their Renilla luciferase activities. Each symbol indicates the sample from a single mouse, and bars indicate the median values. RLU, relative light units. *, P < 0.05; **, P < 0.001.

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