Immune responses in the “PIV5 naïve” dogs inoculated with rPIV5-H3.

<p>The dog blood samples were collected at 0 and 21 days post infection. 4 HAU of the influenza A virus (A/Udorn/72, H3N2 subtype) were mixed with serially diluted dog sera in 96-well round-bottom plates. The hemagglutination inhibition (HAI) titer was scored as the reciprocal of the highest dilution antiserum that completely inhibits hemagglutination. The graph shows the mean value of duplicate wells for each dog. The limit of detection of the HAI titer (10) is indicated.</p

PIV5 antibodies in humans.

<p>45 human serum samples were obtained from 18–50 year old healthy individuals. (A) Comparison of anti-PIV5 and anti-MuV antibody levels. ELISA was performed on plates coated with purified PIV5 or purified MuV with sera serially diluted. PIV5 or Mumps virus specific ELISA OD₄₅₀ values were shown at 320-fold dilution for each human serum sample. (B) Titers of neutralizing antibody against PIV5 in human sera. Data for the antibody titers were the average value of duplicate wells and presented for each human sample. The white column indicates that the PIV5 nAb titer is less than 10, the limit of detection. The black column indicates that the nAb titer is equal to or higher than 10.</p

Titers of anti-PIV5 antibodies in the PIV5-vaccinated dogs.

<p>Eight dogs which had been vaccinated with live PIV5 were immunized with one dose of 8×10⁷ PFU of rPIV5-H3 viruses in 1 mL or PBS via intranasal route. The dogs were divided into two groups: two dogs received PBS; The remaining six dogs received rPIV5-H3. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and viral neutralization antibody assay (B). Data were presented as average value of duplicate wells. In the neutralization antibody assay, the white column indicates the PIV5 nAb titer is equal to or higher than 10.</p

Titers of anti-PIV5 antibodies in dogs without PIV5 exposure.

<p>Eight PIV5 naïve dogs were immunized with one dose of 8×10⁷ PFU of PIV5 or rPIV5-H3 viruses by intranasal route. The dogs were divided into two groups: PIV5-infected dogs and rPIV5-H3-infected dogs. Blood samples were collected at 0 and 21 days post infection for ELISA (A) and virus neutralization antibody (nAb) assay (B). The grey columns indicate that the PIV5 nAb titer is less than 10, the limit of detection in this assay. The black columns indicate that the nAb titer is equal to or higher than 10.</p

Replication of PIV5 in dogs without PIV5 exposure.

<p>The nasal swabs of dogs were collected at 3 and 5 days post infection, and placed into a vial containing 0.5 mL of DMEM with 2% FBS. (A) Detection of virus with RT-PCR. (B) Detection of virus with plaque assay. Swab samples were examined by plaque assay on BHK21 cells. Two replicates for each serially diluted swab sample (1∶10⁰ to 1∶10²) were used in the assay.</p

Replication of PIV5 in dogs with prior PIV5 vaccination.

<p>The nasal swabs of dogs were collected at 3 and 5 dpi. Detections of virus were performed the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050144#pone-0050144-g002" target="_blank">Fig. 2</a>. (A) RT-PCR and (B) Plaque assay.</p