Measurement of antibody binding to CCHF virus Gn protein from mice immunised with MVA-GP.

<p>The absorbance readings from an ELISA assay using the Gn protein as antigen were plotted from all individual donor animals (n = 36). A control antibody preparation from a mouse hybridoma cell line developed against the Gn protein was used as a positive control. The error bar denotes the mean and standard error. The dotted line represents the cut-off level based on mean absorbance + 2 standard deviations of unvaccinated guinea pig control sera.</p

Normalised viral load analysis of CCHFv RNA by RT-PCR.

<p>A129 mice vaccinated with MVA-1974, MVA-GP or which received sera, CD3⁺ T cells, or both from MVA-GP immunised animals were challenged with CCHFv either 14 days after the booster vaccination or 1 day after transfer of immune mediators. Four days post-challenge, three randomly selected animals from each group were killed humanely and analysed by RT-PCR for levels of CCHFv gene expression (normalised to mouse HPRT gene expression). Each point represents the mean value of triplicate measurements in an individual animal. Lines show mean values and error bars denote standard error.</p

Severity of microscopic lesions and presence of viral antigen in tissues from vaccinated and recipient mice 3 days post-challenge with CCHFv.

<p>Severity of microscopic lesions and presence of viral antigen in tissues from vaccinated and recipient mice 3 days post-challenge with CCHFv.</p

Normalised viral load analysis of CCHFv RNA by RT-PCR.

<p>A129 mice vaccinated with MVA-1974, MVA-GP or which received sera, CD3⁺ T cells, or both from MVA-GP immunised animals were challenged with CCHFv either 14 days after the booster vaccination or 1 day after transfer of immune mediators. Four days post-challenge, three randomly selected animals from each group were killed humanely and analysed by RT-PCR for levels of CCHFv gene expression (normalised to mouse HPRT gene expression). Each point represents the mean value of triplicate measurements in an individual animal. Lines show mean values and error bars denote standard error.</p

Survival of A129 mice vaccinated with MVA-1974, MVA-GP or which received sera, CD3⁺ T cells, or both from MVA-GP immunised animals after challenge with CCHF virus.

<p>Animals were challenged with 200 ffu of CCHF virus 14 days after the booster vaccination or 1 day post-transfer of sera, CD3⁺ T cells, or both. Six animals from each group were used to assess survival post-challenge.</p

Weight changes, temperature changes and clinical signs of A129 mice vaccinated with MVA-1974, MVA-GP or which received sera, CD3⁺ T cells, or both from MVA-GP immunised animals after challenge with CCHF virus.

<p>(A) Weight changes were assessed as percentage change compared to the day of challenge. (B) Temperature changes were analysed as the °C difference compared to the day of challenge. (C) Clinical score was a numerical value based on signs recorded each morning of the study. Symbols show the mean value from all surviving animals from each group alive at that time (n = 6 per group challenged with CCHF virus) and error bars denote the standard error.</p

Summary of A/Cal infectivity in nasal washes.

<p>Panel (a) shows ferrets infected with A/Cal on day 0 and treated with 300 µg 244 DI virus (▪) or infected and treated with 300 µg inactivated 244 DI virus (▴); another group was not infected but treated with 300 µg of active 244 DI virus (•). A standard preparation of A/Cal virus was used to normalise titrations carried out on different days. These varied by less than 4-fold. The dotted line is the limit of sensitivity of the assay (1.92 log₁₀ FFU/ml). Significant reduction in infectivity (by a two-tailed Mann-Whitney U test) in ferrets treated with 244 DI virus is denoted by **. Panels (b) and (c) show details of the statistical analysis on day 2 and 3, respectively.</p

Changes in weight of ferrets over the course of infection with A/Cal.

<p>Shown is the mean group body weight changes in A/Cal influenza virus-infected ferrets treated with (a) 300 µg 244 DI virus (▪) or inactivated 244 DI virus (▴), (b) 30 µg 244 DI virus (▪) or inactivated 244 DI virus (▴). (c) Shows the weight changes in ferrets inoculated with saline (○) or treated with 300 µg of active 244 DI virus (•). Data are expressed as a percentage change compared to the group average weight at day 0. The statistical significance of body weight changes on any one day was determined by a one tailed unpaired t-test and is indicated by an asterisk (p≤0.05).</p

244 DI virus (244) RNA is amplified in nasal washes by A/Cal.

<p>Ferrets were infected with A/Cal on day 0 and treated with 244 DI virus or inactivated 244 DI virus. Levels of 244 DI RNA were determined by quantitative RT-PCR. Mean 244 RNA copy numbers for each ferret group (n = 5) are plotted. Panel (a) shows ferrets that were infected with A/Cal influenza virus and treated with 300 µg 244 DI virus (▪), or 30 µg 244 DI virus (▴), or 300 µg (i) inactivated 244 DI virus (▾), or 30 µg (i) inactivated 244 DI virus (⧫). Panel (b) shows non-infected ferrets that were given 300 µg 244 DI virus (□), or diluent (▵). The dotted line shows the limit of detection. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049394#pone.0049394.s001" target="_blank">Figure S1</a> gives details for individual animals.</p

Statistical analysis of summed clinical signs for each day in ferrets re-challenged with A/Cal.

<p>The group that previously experienced A/Cal+300 ug 244 DI virus (▪) is compared with the group that previously experienced only saline (▴). The p value was determined using a one tailed Mann-Whitney U test.</p