Ratio of CD8⁺ INF-γ⁺ cells in splenocytes in response to M2-1, F, and NP peptides.

<p>Cotton rats were infected with the RSV Long strain and splenocytes were obtained four days after the first, second, third, and fourth infections. Data are shown as the average of three (at the first and second infections) to six (at the third and fourth infections) animals. The number of cells was counted in gate. Data show the ratio of the number of cells stimulated with the antigen divided by the number of cells incubated in RPMI buffer. Bars represent means ± SE.</p

Histopathological findings of lung tissues following infections with the RSV Long strain.

<p>(A) The results of hematoxylin-eosin (HE) staining and immunostaining are shown. Lung tissues were obtained from cotton rats inoculated with the supernatant of the HEp-2 cell homogenate through an intranasal route as a negative control (a). Lung tissues were obtained four days after the first, second, third, and fourth infections and the results of HE staining are shown (b-e). Lung tissues were stained with a four-clone blend of monoclonal antibodies against the RSV P, F, and N proteins and anti-mouse IgG conjugated with HRP (f-i). Each panel shows the magnification of 1:200. (B) Lung tissues infected with the Long strain were scored. Lung tissues were sectioned and stained with HE. Six random fields per group were scored for histopathology based on the following criteria: narrowing alveolar space, thickness of alveolar walls, destruction of bronchial epithelial cells, peribronchial infiltration of inflammatory cells, mucus production, and bleeding. Bars represent means ± SE.</p

Experimental schedule for repetitive respiratory syncytial virus infections.

<p>Cotton rats were infected with the Long strain (1×10⁶ pfu/rat). Three cotton rats each were sacrificed 4 days after the first, second, third, and fourth infections. The third and fourth infection were analyzed twice. In order to examine immune responses to subgroup A and B, twelve cotton rats were prepared and divided into four groups. Six rats were challenged with RSV/A/Tokyo/2012 or RSV/B/Tokyo/2012 one month after the second infection, and sacrificed the groups of wild-type A-3 and B-3. The other rats were challenged one month after the third infection, and sacrificed. Wild-type A-3, B-3, A-4, and B-4 are showing a third and fourth infection with RSV/A/Tokyo/2012 or RSV/B/Tokyo/2012.</p

Recovery of the infectious RSV Long strain from lung tissues.

<p>Three cotton rats were infected with 1.0×10⁶ PFU of the RSV Long strain. Virus infectivity was monitored in lung homogenates, and RSV infectivity was shown as PFU per 1 gram of lung tissue. B.D.L indicates below the detection limit.</p

Neutralizing antibody responses against RSV.

<p>(A) Serum samples were obtained 4 weeks after the first, second, and third infections. Neutralizing (NT) antibody responses were examined using the 50% plaque reduction assay with the Long strain. Bars show the average (pre and first infection: n = 3, second and third infections: n = 6 rats per group). Three serum samples were obtained 4 weeks after the first infection, and six samples were collected after subsequent infections. (B) Serum samples obtained one month after the third infection were used. Serum samples were mixed with RSV/A/Tokyo/2012 or RSV/B/Tokyo/2012, incubated at room temperature, and inoculated on Vero cells. They were stained with polyclonal antibodies against RSV conjugated with HRP. Data are shown as the average of three animals per group.</p

Antibody responses of EIA and NT antibodies against JEV.

<p>Three cotton rats were immunized with a recombinant MVAIK/JEVprM-E virus and reimmunized eight weeks after the first immunization. Serum samples were obtained after 1, 3, 5, 8, and 12 weeks. EIA and NT antibodies against JEV were measured.</p

Construction strategy of recombinant cDNA.

<p>The PrM-E region was amplified from the Beijin strain of Japanese encephalitis virus and cloned into the P/M junction of pMVAIK, using <i>Nco</i> I and <i>Not</i> I restriction enzyme sites.</p

Protein expression of JEV of culture fluid and purified measles virus particles.

<p>Vero cells were infected with a recombinant virus, and cell lysates, culture supernatants, and cell culture fluids were then immunoprecipitated using a polyclonal antibody against JEV. They were subjected to Western blotting (4A). Recombinant virus particles were purified through sucrose discontinuous density gradients. Fractions 1, 2, and 3 were collected and subjected the Western blotting. Polyclonal antibodies against measles virus (left panel) and monoclonal antibodies against the JEV E protein were used (4B).</p

Expression of JEV E and measles N proteins.

<p>Monoclonal antibodies against the JEV E and measles N proteins were used, and visualized by anti-mouse antibodies conjugated with FITC or rhodamine.</p

Virus growth in Vero cells at different temperatures of 33, 35, 37, and 39°C.

<p>Virus infectivity was assayed as TCID₅₀ in Vero cells and each bar represents the mean ± 1.0 SD.</p