Abstract
Background
Duck enteritis virus can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of duck enteritis virus (DEV) vaccine strain pDEV-EF1 has been constructed in our previous study. To visually studying DEV, a double-labeled recombinant virus have been constructed.
Methods
based on pDEV-EF1, a recombinant mutated clone pDEV-UL35(c)CFP-gC(c)mRFP which carries a red fluorescent protein(mRFP) gene attached to the viral envelope protein gC, combined with a cyan fluorescent protein (CFP) gene fused to the viral capsid UL35 gene was constructed by two-step Red/ET recombination and the recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Then protein expression were detected by Western blot analysis, and subcellular location of gC and UL35 were observed by confocal microscopy. Viral morphogenesis were observed by transmission electron microscopy (TEM).
Results
The recombinant virus rDEV-UL35(c)CFP-gC(c)mRFP was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed UL35-CFP and gC-mRFP are both expressed in fusion forms in rDEV-UL35(c)CFP-gC(c)mRFP-infected CEFs, and subcellular location study showed gC-mRFP was mainly localized in whole cell at 36 h.p.i. and 48 h.p.i.; and then mostly migrated to cytoplasm after 60 h.p.i.; UL35-CFP was localized in nucleus in all stages of virus infection. Transmission electron microscopy indicated that viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells. However,mature C capsids was less in rDEV-UL35(c)CFP-gC(c)mRFP-infected cells than rDEV-dEF1GFP and rDEV-gC(c)mRFP-infected samples.
Conclusions
This study has laid a foundation for visually studying localization, transportation of DEV capsid protein and envelope glycoprotein, as well as virus assembly, virion movement and virus-cell interaction.
Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies
