Figure 1.
Molecular surface representations of the CMV virion.
The whole molecular surface is colored in gray (A). External coat protein loops are colored as follows (B): loop 1 (76–83 aa) is green, loop 2 (113–118 aa) is red, loop 3 (129–136 aa) is blue, loop 4 (156–163 aa) is magenta and loop 5 (193–199 aa) is yellow. The PCV2 epitope insertion points are indicated in panel C. Ser 131 and 132 are represented as orange beads. Panel D is the superposition of B on C.
Figure 2.
CMV and PCV epitopes on the virion surface.
Spatial localization of the epitopes on the modified CMV CP trimer (A, B) and on the predicted PCV2 pentamer surface (C, D). The epitope (PCV2_224-233) was inserted after the 131 aa position. The CMV subunits A, B and C are cyan, pink and gold, respectively. The epitope is illustrated in licorice representation. The basic amino acids are blue, acidic amino acids are red, the non-polar amino acids are gray and the polar amino acids are colored in green. Plan view of the external surface of the predicted PCV2 CP pentamer (C) and the side view of the pentamer showing the outer surface and the inner surface part (D). External PCV2 capsid protein epitope colors: PCV2_37-43 is ice cube, PCV2_90-96 is mauve, PCV2_126-145 is cyan, PCV2_169-186 is pink and PCV2_224-233 is colored with the above used color schemes.
Figure 3.
Visualization of epitope insertion points in CMV.
Localization of stable epitope insertion sites G83/S84 (blue), G131/S132 (red) and D176/I177 (green) in CMV. The first two sites are on the external surface of the virion while the third is located towards the inside of the virion (A). The CMV subunits A, B and C are cyan, pink and orange, respectively. The symmetrical distribution of the inserted epitope (red) is visible on the Van der Waals representation of the modified CMV virion surface (B).
Figure 4.
X-ray structure of the PCV2 virion (PDB ID code: 3R0R).
The last seven residues are not present in the X-ray structure but it is well visible that the C-terminal tail (red) of the PCV2 capsid protein is located at the edge of the CP pentamer.
Figure 5.
Northern blot analysis of accumulated viral RNAs in systemically infected leaves of N. clevelandii Gray plants 14 days after the inoculation. The hybridization probe was specific to RNA3 of CMV. Ethidium bromide-stained rRNA from the same volume of each sample is shown below each lane. Lane 1: mock inoculated, 2: R-CMV, 3: pR3/131-PCV224-233, 4: pR3/131-PCV126-145.
Figure 6.
Detection of PCV2 specific antibodies.
Indirect immune fluorescence test results of sera from mice immunized with recombinant (A) or wild type (B) CMV, detected in PCV2 capsid expressing baculovirus infected cells. The pictures show the results obtained with 1∶40 serum dilution of both antibodies.
Figure 7.
Anti-PCV2 antibody response of pigs.
Bars indicate the change of the antibody titers of pigs immunized by recombinant CMV-PCV2_224-233 (group 1) or inactive PCV2 (group 2) or mock (group 3) and challenged with PCV2, as measured by indirect immune fluorescence test.