Figure 1.
Generation and growth of rMVKSEGFP.
(A) Plasmids generated after RT-PCR, cloning and sequencing of MV RNA isolated from MVKS-infected PBMC. pMVKS is a full-length plasmid containing the complete antigenome of MVKS and pMVKSEGFP was modified by the insertion of an ATU at the promoter proximal position containing the ORF encoding EGFP. (B) rMVKS and rMVKSEGFP were rescued from Vero-SLAM cells and passaged in B-LCL. Fluorescence microscopy confirmed high levels of EGFP expression in rMVKSEGFP infected cells. (C) Growth curves of MVKS, rMVKS, rMVKSEGFP and rMVIC323EGFP in human B-LCL. Virus was harvested 24, 48, 72 and 96 hours post infection, CCID50 was determined in an endpoint titration test. Measurements shown are averages of triplicates ± SD. Key: h.p.i.: hours post infection.
Figure 2.
Early rMVKSEGFP replication in the respiratory tract.
(A) Virus isolation performed from nose and throat swabs (left two panels), and from BAL cells (right panel). Each symbol represents an individual animal, bars indicate the geometric mean. Key: VI: virus isolation; d.p.i.: days post infection. (B) Live cell confocal microscopy performed on agarose-inflated lung slices from animals on 2 and 3 d.p.i. EGFP+ cells are shown in green, DAPI was used to counter stain nuclei (blue). Three images were collected, labeled i, ii and iii. Panels iia and iib show infected cells in one image from different orientations. Matching 3D-videos for (Bi, Bii and Biii) are available as supporting data.
Table 1.
Dissemination of MV in tissues during early stage of infection.
Figure 3.
Systemic rMVKSEGFP replication.
(A) MV load in PBMC and LN. The left panel shows virus isolations performed from PBMC, each symbol represents an individual animal, bars indicate the geometric means. The right panel shows the presence of MV genome in the axillary LN (crosshairs, geometric mean in blue) and in the tracheobronchial LN (triangles, geometric mean in red). Key: VI: virus isolation; RT-PCR: real-time reverse transcriptase PCR; d.p.i.: days post infection. (B) Detection of EGFP+ cells by flow cytometry from the retropharyngeal LN (left) and the tracheobronchial LN (right) on 2, 3, 4 and 5 d.p.i. Data are shown as dot plots of FL-1 (EGFP) versus FL-2 (empty channel), generated with BD FACSDiva software. In these plots autofluorescent cells usually appear on a diagonal line as they cause comparable signals in both channels. The EGFP-positive events were gated as indicated by the curvilinear line. Data of a representative animal are shown on each time point. Numbers of EGFP+ cells per million total cells are shown in each plot. (C) Representative example of macroscopic EGFP detection at 5 d.p.i. Arrow indicates the infected tonsillar tissue expressing EGFP. Key: Tg: tongue; Tn: tonsil; L: larynx.
Figure 4.
Characterization of MV infection in BALT structures.
(A) H&E staining on lung slice from an animal euthanized on 3 d.p.i.. The number of EGFP+ foci was extremely low, the boxed area (Ai) is a BALT which was the only area on the section where EGFP+ cells were present. (Aii) shows a serial section stained with anti-GFP (black) to detect the presence of virus (see also Figure S3, annotated immunohistochemical and H&E annotated pathology scans). (B) Indirect dual immunofluorescence of the infected BALT structure, showing the presence of T-lymphocytes (CD3), DC or macrophages (CD11c, mac387) and B-lymphocytes (CD20) within the BALT. The BALT is lined by a layer of cytokeratin-positive epithelial cells, and has a blood vessel with CD31-positive endothelium running through it transversely. (C) Higher magnifications of dual immunofluorescence within the BALT indicates the presence of MV-infected T-lymphocytes (CD3), DC or macrophages (CD11c) and B-lymphocytes (CD20), Double positive cells are indicated by arrows. In panel (B) and (C), EGFP+ cells are shown in green, cell-type specific staining is shown in red. DAPI was used to counter stain nuclei in blue. (D) Dual immunofluorescence performed on uninfected BALT region. Dual labelling with cytokeratin (green) and CD3, CD11c or CD20 (red) showed that T-lymphocytes, B-lymphocytes and DC or macrophages are present in very close proximity or in direct contact with the alveolar or bronchiolar lumen (asterisks). Single colour images for (C) are available as supporting data (figure S2).
Figure 5.
Dissemination of MV into the lymphoid organs via blood vessels.
(A, B) H&E staining (left panel) and EGFP staining (right panel) on serial sections of tonsils at 4 d.p.i. (A) and 5 d.p.i. (B). Asterisk denote the proximity of venules to MV-infected cells. (C) Dual labeling of EGFP (green) and the endothelial marker CD31 (red) performed on the tonsils from animals euthanized 5 d.p.i. The left panel shows MV-infected cells in close proximity to CD31+ endothelial cells of venules (arrows), the right panel shows an MV-infected cell migrating through the wall of the venule (arrow). DAPI was used to counter stain nuclei in blue. Single color images for (C) are available as supporting data (figure S2).