Origin and sampling date of analyzed HBV genotype S full-length and S-gene sequences.

<p>Origin and sampling date of analyzed HBV genotype S full-length and S-gene sequences.</p

Phylogenetic analyses of all available HBV/E S-gene and full-length sequences.

<p>Analyses of S-gene (a) and full-length sequences (b) were performed using the GTR+G+I model with geographic information. Branching and roots of strains from individual countries are indicated by colors. Clusters with strains sampled in the same country and during the same year are collapsed.</p

Fusion activity of SSPE MeV-H.

<p>A, Vero, Vero/hSLAM, CHO/SLAM, CHO/CD46, and CHO/nectin-4 cells were cotransfected with DNA expression plasmids for the vaccine MeV-F and the MeV-H indicated at the top. At 24 hours, cells were stained and micrographs were taken at ×4 magnification. B, Fusion activity observed in Vero/hSLAM cells was quantitated by counting the number of nuclei in randomly chosen syncytia. Statistical probabilities, calculated by ANOVA, are indicated. C, Surface expression of the SSPE MeV-H proteins. Levels of cell surface expression were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192245#pone.0192245.g001" target="_blank">Fig 1D</a>.</p

Characteristics of subacute sclerosing panencephalitis cases.

<p>Characteristics of subacute sclerosing panencephalitis cases.</p

Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses

<div><p>Subacute sclerosing panencephalitis (SSPE) is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV) that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H) found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3–4 months, SMa79), average (3.5 years, SMa84), and long (18 years, SMa94) disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs) or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4–dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79) were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79) showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a pathogenetic mechanism in SSPE.</p></div

Median Joining Network of HBV/E S-gene sequences.

<p>Pie charts represent sequence variants at the nodes, with colors indicating the country of sampling of individual sequences, the sizes reflecting the frequencies of the corresponding variants.</p

Bayesian skyline plot showing the epidemic history of the HBV/E S-gene dataset.

<p>The plot indicates the median estimate of the effective population size, with the 95% highest posterior density indicated in blue. The applied timeframe ranges between the most recent sampling date and the calculated 130 years of evolution from the most recent common ancestor (MRCA), as calculated in the HBV/E full-length analysis.</p

Phylogeographic spread of HBV/E.

<p>The snapshots represent the geographic and temporal spread of HBV/E for which at least the S-gene and the spatial and temporal sampling information were available. A mutation rate of 7×10⁻⁵ s/s/y with the GTR+G+I model with geographic information was used. Spread analysis by the SPREAD software was visualized using Map Resources.</p

Syncytium formation of virally expressed MeV-Hs.

<p>Vero/hSLAM cells were infected with recombinant MeV expressing the indicated MeV-H. Syncytia size was measured 24 hours post transfection. Statistical significance (*<i>P</i> < .05; ***<i>P</i> < .001) was calculated by one-way ANOVA with post-hoc Tukey multiple comparisons. Differences in syncytia formation were significant between MeV-H from SSPE cases and genotype A when other wild-type genotypes were excluded from the analysis (A vs B). C, Protein composition of virus stocks. Recombinant MeVvac2(GFP)N (10⁴ plaque-forming units) possessing SSPE-specific MeV-H protein were immunoblotted with antibodies against MeV-N, MeV-H (anti-cytoplasmic and anti-globular head), MeV-F, and GFP proteins. Protein intensity was determined using a ChemiDoc Imaging System (Bio-Rad), with the MeV genotype A, set to 1, used as the comparator. Note that similar levels of MeV-H are detected when anti-cytoplasmic tail–specific antibodies are used but not when antibodies against the variable MeV-H globular are used.</p

Glycosylation and Oligomerization state of SSPE MeV-H.

<p>A, Electrophoretic mobility of PNGase F-treated or untreated SSPE MeV-H proteins. Cytoplasmic extract of Vero cells transfected with plasmid DNA encoding MeV-F protein from genotype A and different MeV-H forms (genotype A; genotype F, patients SMa79 and SMa94; and genotype C1, patient SMa84) were treated with or without PNGase F before they were immunoblotted with the indicated antibodies. MeV-F served as a double control for transfection and gel electrophoresis. In the presence of PNGase F, the electrophoretic migration pattern is not affected by glycosylation status. The apparent molecular mass is indicated above the corresponding band for the untreated MeV-H. MeV-H genotype C1 lacks a potential N-linked glycosylation site (PNGS) at N200 and shows a downward band shift compared with MeV-H genotype A and F (SMa94) proteins. MeV-H F (SMa79) cannot be detected by anti-MeV-H cytoplasmic tail antibodies, but the anti-MeV-H globular head BH195 antibody revealed a similar downward band shift regardless of the presence of N-linked glycans. B, Glycosylation of MeV-H mutants. MeV-H encodes 5 PNGS at N168, N187, N200, N215, and N238, but only the first 4 have been shown to be glycosylated (theoretical vs experimental glycosylation). An N200D mutation would destroy an active PNGS, whereas F180S/L181P and D416N would create additional PNGSs. A downward shift is observed for N200D, whereas an upward shift is seen for the D416N mutant. No changes in electrophoretic mobility were observed for the F180S/L181P mutant. After removal of N-linked glycans, all proteins migrate equally. D416N shows an additional band likely corresponding to a +1 PNGS. C, Oligomerization state of SSPE MeV-H proteins. Cytoplasmic extracts were resolved under nonreducing conditions and immunoblotted with anti-MeV-H_cyt. The different oligomeric states are indicated. D, Surface expression of the mutant MeV-H proteins. Cell surface expression was monitored by flow cytometry after transfection with pCG-MeV-H (genotype A) expression plasmid and the mutants used in panel B. Empty pCG plasmid was used as negative control. MeV-H protein surface expression was detected with anti-MeV-H BH026 antibody followed by goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594.</p