Fig 1.
DTMUV reverse genetics strategy.
(A) Cloning design. Total RNA was extracted from the DTMUV FX2010-180P strain and reverse-transcribed into cDNA. Six cDNA segments were amplified using High Fidelity DNA polymerase pfx and inserted into plasmid vectors. The three 3’ cDNA segments were sub-cloned into plasmid p3656-10991. To generate the full-length DNA corresponding to the entire viral genome, a series of PCRs was performed using using pfu Ultra II Fusion HS DNA polymerase with the recombinant plasmids as templates. (B) Genome schematics of rFX2010-180P and rFX2010-180P-EGFP. rFX2010-180P-EGFP expresses eGFP from an IRES located between the NS5 gene and the 3’ UTR.
Table 1.
Primers used in this study.
Fig 2.
Indirect immunofluorescence microscopy of DF-1 cells infected with the rescued rFX2010-180P virus as detected using the monoclonal antibody 1F5.
(A) rFX2010-180P-infected cells. (B) Uninfected cells.
Fig 3.
DF-1 cells were infected with rFX2010-180P or FX2010-180P at multiplicities of infection of 0.1 and the supernatants were titrated on DF-1 cells at the indicated time points.
Fig 4.
Six 8-week-old shelducks were inoculated with 105.5 TCID50 of rFX2010-180P or FX2010-180P intramuscularly. Antibodies generated against DTMUV in infected ducks were quantified by performing blocking ELISAs using serum obtained at 4–7 dpi.
Fig 5.
DF-1 cells were infected with rFX2010-180P-EGFP and observed using fluorescence microscope. Fluorescence is shown as a function of viral passage number and in comparison with uninfected cells.
Fig 6.
Comparison of rFX2010-180P and degenerated rFX2010-180P-EGFP.
(A) Sequence differences of degenerated rFX2010-180P-EGFP (a) and rFX2010-180P (b). (B) RNA structure prediction of r-FX2010-180P (a) and degenerated rFX2010-180P-EGFP (b) using RNAstructure 5.3 software. (C) Growth kinetics of rFX2010-180P and degenerated rFX2010-180P-EGFP were compared in DF-1 cells.