Figure 1.
In vitro characteristics of recombinant viruses.
(A) Viral growth kinetics in Vero cells infected with viruses at an MOI of 0.01 analyzed by TCID50 assay. Data from three independent experiments are shown. (B) Plaque morphology of viruses on Vero cells.
Figure 2.
Expression of GFP and VP1 in cultured cells.
(A) Vero cells infected with rCV-GFP or transfected with pEGFP-N1 (control), showing green fluorescence (GFP) and red fluorescence (viral protein, probed with anti-enteroviral VP1 antibody). (B) GFP expression from rCV-GFP examined at passages 2 to 10 by immunoblotting using anti-GFP antibody, anti-enteroviral VP1 antibody, or anti-β-actin antibody, with pEGFP-N1 and CVB3 as controls. Scale bar 50 µm.
Figure 3.
GFP expression from rCV-GFP in infected mice heart.
Expression of GFP (green fluorescence) in heart at day 2 (A), day 4 (B), and day 6 (C) post-infection. Higher magnification of inset showing GFP (D), viral capsid protein in red fluorescence (E), and their co-localization (F). Scale bar, 100 µm.
Table 1.
Comparison between wild type and attenuated CVB3 genomes.
Figure 4.
Virulence attenuation of recombinant viruses in mice.
Mice were inoculated with 105 TCID50 of viruses and sacrificed on scheduled days. (A) Mice in CVB3-WT group showing poor general condition and skin ulceration from days 8 to 14 post infection. (B) Hematoxylin and eosin histology of hearts infected with CVB3-WT, rCV-CS, or rCV-GFP, showing myocardial inflammation in CVB3-WT infected heart (arrows).
Figure 5.
Titers of viruses in tissues of mice.
The titers of each virus in the pancreas (A), heart (B), and liver (C) were determined by TCID50 assay on days 2, 4, and 6 post-infection.
Figure 6.
Schematics of recombinant CVB3 expression vector.
(A) Primers used to generate sub-genomic CVB3 fragments (see Table 2) and the assembly strategy of the complete genome of CVB3. (B) Elaborate map of a duplicated 2Aproteinase cleavage sites and two unique cloning sites (ClaI and StuI) in pCV. (C) Relative position of an exogenous gene GFP in the viral genome of pCV-GFP. During viral RNA translation and polyprotein proteolytic processing, GFP will be excised from the mature protein at 2Aproteinase cleavage sites. Abbreviations: T7P, T7 RNA polymerase-dependent promoter; NTR, Non-translated region of the CVB3 genome; VP, Viral capsid protein.
Table 2.
PCR Primers used in this study.